PulseNet Quality

Assurance/Quality
Control (QA/QC) Manual
 Quality Assurance / Quality Control Manual for the Standardized

Pulsed-Field Gel Electrophoresis Technique Used by

CDC PulseNet Laboratories Foodborne Disease Surveillance

(Version 2.0 for the Centers for Disease Control and Prevention)

May 9, 2005 1
 For internal use by CDC PulseNet participating laboratories.
Please do not distribute without permission.
Send all corrections, comments and questions to
[email protected]

May 9, 2005 2
 TABLE OF CONTENTS

I. PREFACE

II. INTRODUCTION

III. OBJECTIVE

IV. SCOPE

V. DEFINITIONS

VI. QUALITY ASSURANCE PROGRAM

A. GOALS AND OBJECTIVES

B. ORGANIZATION AND MANAGEMENT

C. POLICY FOR PERFORMING PFGE

D. PERSONNEL TRAINING AND CERTIFICATION

E. SPECIMENS

F. ANALYTICAL PROCEDURES

G. DOCUMENTATION

H. EQUIPMENT: CALIBRATION AND MAINTENANCE

I. CERTIFICATION

J. PROFICIENCY TESTING

K. REVIEW

L. CHANGES TO THE STANDARDIZED PFGE PROTOCOL

M. VALIDATION OF NEW METHODS

N. SAFETY

May 9, 2005 3
 Quality Assurance Standards for Pulsed-Field Gel
Electrophoresis (PFGE)
Testing for the CDC PulseNet Laboratories
I. PREFACE

The objective of this document is to provide CDC laboratories with a set of quality control and
quality assurance standards to be followed when subtyping foodborne bacterial pathogens with
any of the approved standardized protocols. Strict adherence to standardized procedures is
essential to the operation of PulseNet and to ensure that the data submitted to the PFGE National
database is of the highest quality possible. All PulseNet CDC laboratories must comply with the
following quality assurance standards in order to continue to participate in PulseNet.

The PulseNet USA Task Force is committed to assisting all PulseNet participating laboratories
(PulseNet USA and PulseNet International) in achieving the highest quality of data before
submitting patterns to the databases in the respective countries or regions. Part of that
commitment consists of the development and implementation of standardized laboratory
protocols and software analysis parameters that would enable the exchange and comparison of
molecular fingerprinting data between laboratories. It is in this context that the PulseNet Task
Force presents this document as a template to assist all PulseNet USA and PulseNet International
participating laboratories in the development of their own QA/QC manual and program. The
standards presented herein were established specifically for PulseNet laboratories at CDC with
the understanding that most of the following standards will apply, within varying degrees, to all
laboratories participating in PulseNet. We encourage all PulseNet laboratories to adopt these
standards whenever possible and to modify them in a manner consistent with the internal policies
or guidelines established by their institution, state or country.

Effective Date: These standards shall take effect May 9, 2005.

II. INTRODUCTION

This document consists of definitions and standards as they apply to the protocols and
procedures used in PulseNet USA. The standards are quality assurance measures that place
specific requirements on CDC laboratories conducting PFGE analysis with the purpose of
submitting DNA fingerprint patterns/gel images to the PulseNet USA National database(s).
Additional or equivalent measures not outlined in this document may also meet the standard if
submitted and approved by the PulseNet USA Task Force of the Foodborne and Diarrheal
Diseases Branch at the Centers for Disease Control and Prevention.

This document complies with Good Laboratory Practices (GLPs: 21 CFR Part 58) for conducting
nonclinical laboratory studies, which are regulated by the Food and Drug Administration.

This document also satisfies all requirements for the International standard: ISO/DIS 17025
(general requirements for the competence of testing and calibration laboratories).

May 9, 2005 4
Molecular subtyping by PFGE is used for epidemiology and surveillance purposes and the
results are not used for patient diagnosis or treatment. Thus, these procedures are not subject to
the Clinical Laboratories Improvement Amendment (CLIA), 1988. However, every effort has
been made to ensure that this document meets CLIA’88 regulations wherever possible.
Laboratories that report PFGE data directly to physicians, hospitals, etc. must ensure that they
are fully compliant with all CLIA’88 regulations.

The PulseNet USA system encompasses laboratories from the Centers for Disease Control and
Prevention (CDC), Food and Drug Administration (FDA), United States Department of
Agriculture (USDA), State Public Health Departments, and other County and City Public Health
Departments. Certain State Health Departments have been designated as Area Laboratories. The
association of these elements within PulseNet USA is indicated in the Organization Chart in
Appendix A. A map of the PulseNet USA Area Lab regions can be found in Appendix B.

The standardized protocols developed for use by PulseNet laboratories are available from CDC
(E-mail: [email protected]) and are provided within the training course manual “Standardized
Molecular Subtyping of Foodborne Bacterial Pathogens by Pulsed-Field Gel Electrophoresis.”
These protocols also are included in the attached document containing the standard operating
procedures (SOPs). In addition, the protocols can be found on the PulseNet USA Listserv and on
the PulseNet USA website.

III. OBJECTIVE

To institute a quality assurance system to ensure the quality and integrity of the results
obtained with the standardized PFGE techniques used to subtype foodborne bacterial
pathogens.

IV. SCOPE

The standards describe the quality assurance requirements that all the CDC PulseNet
laboratories, defined herein as a facility at CDC in which PFGE analysis is performed for the
purpose of identifying clusters of foodborne illness causing bacteria, must follow to ensure
the quality and reproducibility of the data.

V. DEFINITIONS

The following terms are defined as used in these standards:

1. Administrative review is an evaluation by the PulseNet USA Task Force and/or the
PulseNet USA Steering Committee of the report and supporting documentation for
consistency with laboratory policies and editorial correctness.

2. Analyst is an individual who conducts and/or directs the analysis of all test samples,
interprets data, and reaches conclusions.

May 9, 2005 5
 3. Calibration is the set of operations that establishes, under specified conditions, the
relationship between values indicated by a measuring instrument, system, or material, and
the corresponding known values of that measurement.

4. Certification set is a group of well-characterized strains of a specific organism that are

part of the PulseNet USA certification process. Individuals wishing to become PulseNet
USA-certified must perform PFGE subtyping of these isolates using the appropriate
standardized protocol and/or analyze a TIFF image of these isolates. The certification set
is available from PulseNet USA Task Force upon request.

5. Clearance is the process of review by CDC of all manuscripts, presentations, etc. that
have a CDC employee or contractor as an author.

6. Laboratory is a facility in which sample testing, including PFGE analysis, is performed.

7. Laboratory support personnel are individual(s) who perform laboratory duties but do
not analyze PFGE results.

8. PFGE proficiency test sample is biological material whose DNA type has been
previously characterized by PFGE and which is used to monitor the quality performance
of a laboratory or an individual.

9. Proficiency testing is a quality assurance measure used to monitor performance and

identify areas in which improvement may be needed. Proficiency tests may be classified
as:

(a) Internal proficiency test is one prepared and administered by the laboratory.
(b) External proficiency test (blind) is one that is prepared and administered by the
PulseNet USA Task Force.

10. Pulsed-field gel electrophoresis (PFGE) is a DNA fingerprinting technique that allows
the separation of large DNA fragments (>30 kb) by applying an alternating electric field
between spatially distinct pairs of electrodes.

11. PulseNet International Steering Committee is made up of coordinating officials of the

international PulseNet networks. The committee is chaired by the Chief, Laboratory Unit
of the Foodborne and Diarrheal Diseases Branch at CDC, Atlanta. This Steering
Committee establishes the principles and procedures by which information exchanges
between the PulseNet Networks shall take place. It sets up the rules for sharing of
protocols, standards and other material necessary to obtain comparable DNA
“fingerprint” patterns; sharing of human resources to perform typing and developing and
maintaining methods, protocols, and strain collections; and sharing tasks for the future
development of PulseNet International.

12. PulseNet USA is a network of public health laboratories in the United States that perform
DNA “fingerprinting,” using PFGE, on bacteria that may be causative agents of

May 9, 2005 6
 foodborne disease. The network permits rapid comparison of these fingerprint patterns
through an electronic database maintained at the CDC.

13. PulseNet USA Certification is the process by which a person demonstrates his or her
ability to produce a high-quality gel using the PulseNet USA standardized protocols
and/or the ability to analyze a gel using BioNumerics and the PulseNet USA customized
scripts. A person must be certified for each pathogen for which he/she wishes to submit
PFGE patterns to the PulseNet USA National Database(s).

14. PulseNet USA Steering Committee guides the expansion, improvement, and evaluation
of the PulseNet USA program. Activities of the PulseNet USA network are coordinated
by the PulseNet USA Steering Committee. The PulseNet USA Steering Committee is
chaired by the chief of the PulseNet USA unit of the Foodborne and Diarrheal Diseases
Branch at CDC and is comprised of participants from state and local PulseNet USA
laboratories, FDA, USDA, APHL, and CDC. The Steering Committee meets via
conference call several times during the year.

15. PulseNet USA Task Force is a group of laboratory and database personnel from CDC.
The PulseNet USA Task Force is responsible for the initial review and approval of
documents, development and validation of protocols, and other activities related to
PulseNet USA.

16. Quality refers to freedom from deficiencies.

17. Quality assurance refers to all the activities in which the laboratory is engaged to ensure
that information generated by the laboratory is correct. These activities are to be
implemented in a systematic way to demonstrate that a product or service meets specified
requirements for quality assurance.

18. Quality assurance program is the organizational structure, responsibilities, procedures,

processes, and resources for implementing quality assurance policy.

19. Quality control refers to the process used by the laboratory and other personnel as an aid
to meeting the product or service goals.

20. Quality assurance (QA)/quality control (QC) manual is a document stating the quality
assurance policy, quality assurance program, and quality control practices of an
organization.

21. Real-time PFGE refers to the processing by PFGE, analysis, and submission of PulseNet
USA-tracked isolates to the national databases within 48 hours of their receipt by the
participating lab. The PulseNet USA Task Force and PulseNet USA Steering Committee
recommend that all isolates of Listeria and E. coli O157:H7 be PFGE subtyped in “real-
time.”

May 9, 2005 7
 22. Restriction fragment length polymorphism (RFLP) is generated by cleavage of DNA
with a specific restriction enzyme, and the resulting variation is due to restriction site
polymorphism contained within the DNA used in the analysis.

23. Review is an evaluation of documentation for consistency, accuracy, and completeness.

24. Standard Operating Procedures (SOPs) are the written procedures that detail all
aspects of work in the laboratory or with the database(s). They are written to assure good
laboratory practices are followed in the laboratory and in the analysis of data.

25. Technical supervisor (or equivalent position or title as designated by the laboratory
system) is the individual who is accountable for the technical operations of the
laboratory.

26. Technical review is an evaluation of reports, notes, data, and other documents to ensure
an appropriate and sufficient basis for the scientific conclusions. This review is
conducted by a second qualified individual.

27. Technician is an individual who performs analytical techniques on test samples under the
supervision of a qualified examiner/analyst and/or performs PFGE analysis on samples
for inclusion in a database. Technicians do not evaluate or reach conclusions on typing
results or prepare final reports unless they are also acting as the technical manager or
leader.

28. Validation is a process by which a procedure is evaluated to determine its efficacy and
reliability, and includes:

(a) Developmental validation: the acquisition of test data and determination of

conditions and limitations of a new or novel DNA methodology for use on suspected
outbreak samples.

(b) Internal validation: an accumulation of test data within the laboratory to

demonstrate that established methods and procedures are performed as expected in
the laboratory.

(c) External validation: an accumulation of test data based on the isolates or images
(blinded) provided to the PulseNet USA laboratories by CDC. External validation is
required in order to demonstrate that established methods and procedures perform as
expected.

VI. QUALITY ASSURANCE PROGRAM

Each PulseNet participating laboratory shall establish and maintain a documented quality
assurance program that is appropriate to all the testing activities associated with PFGE or
other PulseNet-related analyses.

May 9, 2005 8
 4.1 The quality assurance/quality control manual shall address the following:

(A) Goals and objectives

(B) Organization and management
(C) Policy for performing PFGE
(D) Personnel training
(E) Specimens
(F) Analytical procedures
(G) Documentation
(H) Equipment: calibration and maintenance
(I) Certification
(J) Proficiency Testing (PT)
(K) Review
(L) Changes to standardized PFGE protocol
(M) Validation of new methods
(N) Safety

A. GOALS AND OBJECTIVES

To standardize the performance of PulseNet USA activities in all CDC participating
laboratories.

B. ORGANIZATION AND MANAGEMENT

STANDARD B.1
The laboratory shall:

(a) Have a managerial staff with the authority and resources needed to discharge their duties
and meet the requirements of the standards in this document.

(b) Have a technical manager or leader who is accountable for the technical operations.

(c) Specify and document the responsibility, authority, and interrelation of all personnel who
manage, perform, or verify work affecting the validity of the DNA analysis.

C. POLICY FOR PERFORMING PFGE

PFGE is not used for diagnostic purposes (i.e., ordered by a physician on a specific patient).
Rather, it is a powerful tool for monitoring and investigating clusters of illness caused by
foodborne bacteria of concern to public health. Each laboratory should formulate its own
statement of policy as to when PFGE is to be performed. Such policy must be consistent
with the “real-time” subtyping guidelines stated in item 21 of the “Definitions” section
(section 3) of this document: laboratories must perform real-time PFGE on all E. coli
O157:H7 and all Listeria monocytogenes isolates received. In situations where real-time
subtyping of Salmonella serotypes, Shigella, and Campylobacter is not possible, laboratories

May 9, 2005 9
 must establish a subtyping policy according to the priorities and resources that still allows for
the early detection of clusters of these organisms.

STANDARD C.1
At the Centers for Disease Control and Prevention, Foodborne and Diarrheal Disease Branch,
PFGE is performed on all isolates 1) submitted by external public health laboratories or
government agencies for that purpose, 2) as requested by CDC epidemiologists investigating
a potential outbreak, 3) as requested by the PulseNet USA National Database Administration
Team for the clarification of PFGE patterns, and 4) for other research purposes.

C.1.1
PFGE needs to be performed in “real-time” to have a significant impact on public
health. All isolates of Listeria monocytogenes or E. coli O157:H7 shall be PFGE
subtyped in “real-time.”

D. PERSONNEL TRAINING AND CERTIFICATION

OBJECTIVE: To standardize the performance of PulseNet activities in all CDC participating

laboratories.

STANDARD D.1
Laboratory personnel shall have the education, training, and experience to carry out required
PulseNet USA activities and responsibilities. The laboratory shall maintain records on the
relevant qualifications, training, skills, and experience of all technical personnel.

STANDARD D.2
The technical supervisor is responsible for technical problem solving of analytical methods
and for the oversight of training, quality assurance, safety, and proficiency testing in the
laboratory.

The laboratory supervisor shall be accessible to provide onsite, telephone, or electronic

consultation as needed.

STANDARD D.3
At least one member of each CDC PulseNet participating laboratory must be trained by
someone approved/recommended by the PulseNet USA Task Force and shall have
successfully completed the certification testing for an organism before being allowed access
to the on-line databases in accordance with the “Standard Operating Procedure for Training
(PNL17 & PND10),” and “Standard Operating Procedure for Certification of PulseNet USA
Personnel (PNQ02).”

This person shall be responsible for training additional staff in their respective laboratory.

STANDARD D.4

May 9, 2005 10
 At least one person from each CDC PulseNet participating laboratory shall attend annual
update meetings and regional meetings when they occur.

E. SPECIMENS
Each laboratory shall formulate its own statement of what is an acceptable specimen for
PFGE.

STANDARD E.1
PFGE is performed on pure cultures. The primary consideration is that the organism be
viable when received. Mixed cultures do not have to be rejected if it is possible to separate
the specific organism in question. Isolates may be from human, animal, produce or animal
food product, or environmental sources. An isolate for which identifiers have been lost or
are in question shall be rejected.

F. ANALYTICAL PROCEDURES

OBJECTIVE: To assure that all reagents and solutions used in the standardized PFGE protocols
are properly prepared and controlled in order to maintain a consistent level of quality.

STANDARD F.1
The laboratory shall have and follow written analytical protocols approved by the PulseNet
USA Task Force and the PulseNet USA Steering Committee.

F.1.1
The laboratory shall use the standard PFGE protocols as outlined in the Standardized
Molecular Subtyping of Foodborne Bacterial Pathogens by Pulsed-Field Gel
Electrophoresis manual (or SOPs PNL03 through PNL06 in this manual), for each
organism to be analyzed by PFGE with the purpose of submitting gel images of
PFGE patterns to the PulseNet USA National Database(s) at CDC.

F.1.2
The procedures shall include reagent preparation, supplies, sample preparation,
extraction protocols, equipment requirements, and appropriate controls that are
standard for DNA analysis and data interpretation.

STANDARD F.2
The laboratory shall use reagents that are suitable for the methods employed. Each
participating laboratory shall be responsible for assuring that reagents and solutions used for
PFGE subtyping are properly prepared, controlled, and stored in order to maintain a
consistent level of quality. The following standards shall apply to all reagents and solutions
used in the standardized PFGE methods.

STANDARD F.3
Good laboratory practices depend upon the use of appropriate chemicals and reagents. An
important aspect of laboratory management is the assurance that materials purchased for

May 9, 2005 11
 testing meet necessary quality control requirements. Stringent specifications for these
materials shall be developed, and adherence to the specifications must be mandatory.

F.3.1
The laboratory shall have written procedures for documenting commercial supplies
and for the formulation of reagents (PNL01 & PNL02).

F.3.1.1
All commercially available reagents shall have a quality assurance certificate
issued by the supplier, a record of which shall be maintained by the laboratory.

F.3.2
Reagents shall be labeled with the identity of the reagent, storage requirements, titer
or concentration, the date of preparation or expiration, and the identity of the
individual preparing the reagent. Deteriorated or outdated reagents and solutions shall
not be used.

F.3.3
Reagent solutions prepared “in-house” shall have their expiration dates established by
the laboratory. A program of periodic testing shall be instituted to determine that
reagents/solutions have not deteriorated.

F.3.4
When establishing reagent preparation as a laboratory function, the methods used
shall be accurately and completely described in writing. There shall be documentation
of each lot prepared, including reagent lots and expiration dates, date prepared,
initials of personnel preparing reagent and expiration date of prepared reagent.

F.3.5
Reagents shall remain free of contamination, either chemical or microbiological.

F.3.6
Proper physical conditions of storage shall be carefully maintained. Reagents shall be
maintained at the temperatures recommended by the manufacturer. Reagents
maintained under refrigerated or freezing temperatures, such as proteinase K and
restriction enzymes/buffers, shall not be removed from the storage conditions until
ready to use. These reagents shall be maintained at the appropriate temperature(s)
following manufacturer’s guidelines while in use, and shall be returned to proper
storage conditions promptly.

F.3.7
The laboratory shall identify critical reagents and evaluate their acceptability prior to
use in sample testing. These critical reagents include but are not limited to:

(a) Restriction enzymes/Proteinase K

(b) In-house/Commercial reagents and buffers

May 9, 2005 12
 (c) S. Braenderup size standard culture/all control plugs

STANDARD F.4
The laboratory shall monitor the analytical procedures using appropriate controls and
standards.

F.4.1
The following controls shall be used in PFGE analysis:

F.4.1.1
Quantification standards for adjusting cell suspensions (i.e., McFarland standards,
etc.).

F.4.1.2
A procedure to monitor the pH or conductivity of buffers (TE, TBE, etc.).

STANDARD F.5
The laboratory shall check its PFGE procedures annually or whenever substantial changes
are made to the protocols against an appropriate and available standard reference material.

F.5.1
Authorized revisions to the Standardized Molecular Subtyping of Foodborne
Bacterial Pathogens by Pulsed-Field Gel Electrophoresis manual or SOPs shall be
incorporated into current practice in a timely fashion.

F.5.2
Any changes to procedures that may affect the comparability of gels between
laboratories shall be sent to the PulseNet USA Task Force for review before any gels
are submitted to the PulseNet USA National Database.

STANDARD F.6
The laboratory shall have and follow written general guidelines for the interpretation of data.
(See PFGE standardization manual, section 13).

G. DOCUMENTATION

OBJECTIVE: To provide all personnel with clear instructions of the goals of the PFGE
technique and all the operations needed to fulfill those goals.

Documentation is divided into four levels, as follows:

Level 1. Quality assurance/quality control (QA/QC) manual. This level is covered by the
present document, which establishes all objectives and policies necessary for achieving the
expected level of quality.

May 9, 2005 13
 Level 2. Standard operating procedures (SOPs). Standard operating procedures are the tools
that provide assurance of the quality and integrity of all data generated during studies
utilizing the standard PFGE technique. All PulseNet participating laboratories shall develop
and follow approved SOPs.

Level 3. Standard protocol(s). PulseNet USA has already developed “One-Day (24-28 hour)
Standardized Laboratory Protocols for Molecular Subtyping of Foodborne Bacterial
Pathogens by Pulsed-Field Gel Electrophoresis (PFGE).” The approved protocols specify all
the recommended methods for conducting PFGE of foodborne pathogens and submitting
images/data to the PulseNet USA National Database(s) (PNL03 – PNL07, and PND02).

Level 4. Records, reports, and images. Includes work documents containing all information
related to the receiving and testing of isolates, as well as results and their analysis and
interpretation. These documents shall be developed according to the policies of the “SOP for
Generation of Records and Reports” (PNL08).

STANDARD G.1
Protocols shall be written according to the “SOP for Writing Standard Operating Procedures”
(PNG01), for consistency and ease of use.

STANDARD G.2
The PFGE standardization manual and protocol development are the responsibility of the
CDC. The procedures can be written by CDC personnel, PulseNet participating laboratories,
or by a collaborative effort between these groups.

STANDARD G.3
CDC PulseNet laboratories shall use the most current version of the standardized protocol.
All CDC PulseNet participating laboratories will receive a copy of the most current
authorized version of the PFGE manual, protocols, and other procedures electronically (e.g.,
via the PulseNet USA Listserv). A master of the most recent version is kept at CDC.

STANDARD G.4
Should any CDC PulseNet participating laboratory generate a document considered useful
for other participating laboratories, that laboratory shall send a copy of the document to the
PulseNet USA Task Force for review ([email protected]). After review and approval, the CDC
will distribute the document to all members of PulseNet (USA and International) for their
knowledge and use.

STANDARD G.5
Reports sent to CDC for analysis shall follow the format included in the “Standard Operating
Procedure for the Generation of Records and Reports” (PNL08).

STANDARD G.6
The laboratory shall have and follow written procedures for taking and maintaining notes to
support the conclusions drawn in laboratory reports.

May 9, 2005 14
STANDARD G.7
The laboratory shall maintain all documentation generated by analysts related to the analysis
of samples.

G.7.1
The guidelines for report writing shall include:
(a) Identifier (e.g., outbreak, sporadic case)
(b) Isolate identification number
(c) Source or description of samples examined
(d) Results (including image and pattern number)
(e) Conclusion and/or interpretative statement if appropriate
(f) Date issued
(g) A signature and title, or equivalent identification, of the person(s) accepting
responsibility for the content of the report

G.7.2
The laboratory shall have written procedures for the release of case report
information.

G.7.3
The laboratory shall follow written local laboratory and PulseNet USA guidelines
when dealing with members of the press (PNG03).

G.7.4
The laboratory shall follow written state and federal guidelines for release of
information in response to a Freedom of Information Act request (PNG04).

H. EQUIPMENT: CALIBRATION AND MAINTENANCE

OBJECTIVE: To assure that equipment and devices work properly.

STANDARD H.1
The laboratory shall use equipment suitable for the methods employed. A list of necessary
equipment is provided in section 5b of the PFGE standardization manual and in SOP PNL01
in this manual.

STANDARD H.2
The laboratory shall have a documented program for calibration of instruments and
equipment. Proper instrument operation and maintenance are necessary to assure quality
results.

H.2.1
All CDC PulseNet laboratories are responsible for proper handling, supervision, and
maintenance of equipment in their respective laboratories.

May 9, 2005 15
STANDARD H.3
Equipment requiring calibration shall be calibrated according to the procedures recommended in
the respective manufacturer’s manual(s). If calibration instructions are not included in the
manual, the laboratory shall request written instructions directly from the manufacturer.

H.3.1
Written records documenting the frequency and date of calibration shall be
maintained for each instrument requiring calibration.

H.3.2
New instruments and equipment, or instruments and equipment that have undergone
repair or maintenance, shall be calibrated before being used in sample analysis.

H.3.3
Written records or logs shall be maintained for maintenance service performed on all
equipment.

I. CERTIFICATION

OBJECTIVE: To ensure that all persons submitting patterns to the National Databases have
been adequately trained on the PulseNet standardized protocol(s) for PFGE and/or Gel Analysis
and are producing good quality gels and analysis.

STANDARD I.1
All members of the CDC PulseNet laboratory (PulseNet Methods Development Laboratory)
shall attend CDC-sponsored training, Area Lab training, or customized training (by a
certified instructor) and must have successfully completed the certification testing for an
organism (in gel preparation, TIFF analysis or both), as described in the “Standard Operating
Procedure for Certification of PulseNet USA Personnel” (PNQ02).

I.1.1
This person shall be responsible for training additional staff in their respective
laboratory.

I.1.2
No person may submit patterns to any PulseNet USA national database without
having been TIFF-certified for that organism.

J. PROFICIENCY TESTING

OBJECTIVE: To participate in the ongoing proficiency testing program, coordinated by the

PulseNet USA Task Force and APHL, to ensure that CDC laboratories participating in PulseNet
USA maintain a satisfactory level of performance for PFGE or other molecular subtyping
methods and gel analysis.

May 9, 2005 16
STANDARD J.1
Each certified PulseNet CDC laboratory shall participate in annual external proficiency
testing for PFGE as described in the “Standard Operating Procedure for the PulseNet USA
Proficiency Testing Program” (PNQ04).

J.1.1
Failure to satisfactorily participate in the external proficiency testing will result in
decertification of the laboratory for that specific organism and termination of access
to PulseNet USA’s on-line database(s). Laboratories may continue to submit .tif and
.bdl files via e-mail.

J.1.1.1
Failure to satisfactorily participate means:

J.1.1.1.1
Failure to return proficiency testing results to the PulseNet USA Task Force.

J.1.1.1.2
Two consecutive unsatisfactory proficiency testing results for the same
organism over two separate testing events.

J.1.2
Laboratories decertified for failure to satisfactorily participate or complete the
external proficiency testing shall have to be recertified (and retrained if necessary)
before restoring access to the PulseNet USA on-line database(s).

K. REVIEW

STANDARD K.1
The failure of any component of the standardized protocol (bad reagents, poor standards,
equipment failure, and/or inadequately trained lab personnel) may result in gels unacceptable
for analysis and inclusion in the PulseNet USA national database(s).

K.1.1
The laboratory shall follow written procedures when a failure is detected in any
component of the standardized PFGE protocol.

K.1.2
Failure of a standard shall be treated as a QC failure for purposes of analysis and
review. Gels on which the standard strain is missing or on which the standard strain
does not match the global standard shall not be analyzed. These gels shall be rerun
using a new plug of the standard strain.

May 9, 2005 17
STANDARD K.2
The laboratory shall conduct administrative and technical reviews of all reports to ensure that
conclusions and supporting data are reasonable and within the constraints of scientific
knowledge.

K.2.1
The laboratory shall have a mechanism in place to address unresolved discrepant
conclusions between analysts and reviewer(s).

L. CHANGES TO THE STANDARDIZED PFGE PROTOCOL

STANDARD L.1
Any changes made to the standardized PFGE protocol(s) shall be appropriately documented.
No changes may be made to the standardized PFGE protocol(s) without approval from the
PulseNet USA Taskforce and the PulseNet USA Steering Committee.

L.1.1
Internal validation of such changes shall be performed and documented by the
laboratory.

L.1.1.1
Changes to the protocol(s) shall be tested using known samples. The laboratory
shall monitor and document the reproducibility and accuracy of the procedure
using the appropriate bacterial DNA control(s).

L.1.1.2
The laboratory shall establish and document match criteria based on empirical
data in conjunction with the PulseNet USA Task Force.

L.1.1.3
Material modifications made to analytical procedures shall be documented and
subject to validation testing by at least three additional PulseNet USA-certified
laboratories designated by the PulseNet USA Task Force.

M. VALIDATION OF NEW METHODS

STANDARD M.1
All developmental validation performed shall be appropriately documented.

M.1.1
Internal validation shall be performed and documented by the laboratory.

M.1.1.1
The procedure shall be tested using known samples. The laboratory shall monitor
and document the reproducibility and accuracy of the procedure using the
appropriate bacterial DNA control(s).

May 9, 2005 18
 M.1.1.2
The laboratory shall establish and document match criteria based on empirical
data in conjunction with the PulseNet USA Task Force.

M.1.3
Where methods are not specified, the laboratory shall, wherever possible, select
methods that have been published by reputable technical organizations or in relevant
scientific texts or journals, or have been appropriately evaluated for a specific or
unique application.

N. SAFETY

STANDARD N.1
The laboratory shall have and follow a documented environmental health and safety
program.

REFERENCES:

PulseNet USA Manual (last revised on September 2003), Standardized Molecular Subtyping of
Foodborne Bacterial Pathogens by Pulsed-Field Gel Electrophoresis. Foodborne and Diarrheal
Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia.

PulseNet USA BioNumerics Training Manual (February 2003). Foodborne and Diarrheal
Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia.

May 9, 2005 19
 APPENDIX A
PulseNet USA Organizational Chart

PulseNet USA National

Databases Located at CDC

PulseNet Participating
Area Labs USDA-FSIS FDA CDC Lab
Public Health Labs

States Not States Not

CFSAN CVM ORA Currently
Currently
Running PFGE Running PFGE

May 9, 2005 20
 APPENDIX B

May 9, 2005 21
 APPENDIX C

STANDARD OPERATING PROCEDURES LIST

General Standard Operating Procedures (PNG)

PNG01: Standard Operating Procedure for Writing Standard Operating

Procedures

PNG03: Standard Operating Procedure for Handling Inquiries from Members of

the Media

PNG04: Standard Operating Procedure for Handling Inquiries from Lawyers and
Freedom of Information Act (FOIA) Requests

PNG05: Standard Operating Procedure for Becoming a PulseNet USA

Participating Laboratory

PNG06: Standard Operating Procedure for PulseNet USA Area Laboratory

Responsibilities

Standard Operating Procedures for the PulseNet USA Laboratory (PNL)

PNL01: Standard Operating Procedure for Laboratory Equipment and Supplies

PNL02: Standard Operating Procedure for Preparation of Reagents Used in the

PulseNet USA Standardized PFGE Protocols

PNL03: Standard Operating Procedure for PFGE of Campylobacter

PNL04: Standard Operating Procedure for PFGE of Listeria monocytogenes

PNL05: Standard Operating Procedure for PFGE of E. coli O157:H7, Shigella,

and Salmonella (pending)

PNL06: Standard Operating Procedure for PFGE of Vibrio cholerae (pending)

PNL07: Standard Operating Procedure for the Image Acquisition and Production
of TIFF Files for Data Analysis

PNL08: Standard Operating Procedure for the Generation of Records and

Reports

PNL09: Standard Operating Procedure for Receipt, Shipping, and Storage of

Isolates (pending)

May 9, 2005 22
 PNL10: Standard Operating Procedure for the Use, Inspection, Cleaning,
Maintenance, and Calibration of Equipment

PNL11: Standard Operating Procedure for Maintenance of Pulsed-Field Gel

Electrophoresis Systems

PNL12: Standard Operating Procedure for Maintenance of Dade Microscan

Turbidity Meter

PNL13: Standard Operating Procedure for Waterbath Maintenance and

Cleaning

PNL14: Standard Operating Procedure for the Use, Storage, and Disposal of
Chemicals and Reagents

PNL15: Standard Operating Procedure for Storage and Use of Expired

Reagents

PNL16: Standard Operating Procedure for Evaluation and Correction of Failure

of Any Component of Standardized PFGE Protocol

PNL17: Standard Operating Procedure for Laboratory Training

Standard Operating Procedures for the PulseNet USA Databases (PND)

PND01: Standard Operating Procedure for Computer Equipment and Supplies

PND02: Standard Operating Procedure for Image Analysis Using BioNumerics

Software

PND03: Standard Operating Procedure for PulseNet USA Listserv Access and
Use

PND04: Standard Operating Procedure for Gel Analysis Guidelines

PND05: Standard Operating Procedure for BioNumerics Administrative Duties

(pending)

PND06: Standard Operating Procedure for Obtaining and Updating Organism

Serotype Codes (pending)

PND07: Standard Operating Procedure for Updating and Maintaining Gel

Analysis Software and PulseNet USA Scripts (pending)

PND08: Standard Operating Procedure for Setup and Use of SecurID Key Fob

PND09: Standard Operating Procedure for Authenticating and Connecting to the

Server

May 9, 2005 23
 PND10: Standard Operating Procedure for Database Training

Standard Operating Procedures for the PulseNet USA QA/QC Program (PNQ)

PNQ01: Standard Operating Procedure for TIFF Quality Grading Guidelines

PNQ02: Standard Operating Procedure for Certification of PulseNet USA

Personnel

PNQ03: Standard Operating Procedure for Evaluation of Certification Files

PNQ04: Standard Operating Procedure for the PulseNet USA Proficiency

Testing Program

PNQ05: Standard Operating Procedure for Laboratory In-House Gel

Certification (pending)

May 9, 2005 24
 PulseNet QA/QC Manual

Standards

General Standard Operating Procedures (PNG)

PNG01 Standard Operating Procedure for Writing Standard Operating Procedures
PNG02 Standard Operating Procedure for Processing In and Out of PulseNet
PNG03 Standard Operating Procedure for Handling Inquiries from Members of the Media
Standard Operating Procedure for Handling Inquiries from Lawyers and Freedom of
PNG04 Information Act (FOIA) Requests
Responsibilities for a PulseNet Laboratory and Standard Operating Procedure for
PNG05 Becoming a PulseNet Laboratory
PNG06 Standard Operating Procedure for PulseNet Area Laboratory Responsibilities
Standard Operating Procedure for Sharing Database Information between PulseNet USA
PNG07 and PulseNet Canada
Standard Operating Procedure for Becoming a PulseNet Participating Laboratory and
PNG08 Handling Requests to Become a PulseNet Laboratory
Standard Operating Procedure for Sharing Database Information between PulseNet USA
PNG09 and USDA VetNet (pending)

Standard Operating Procedures for the PulseNet Laboratory (PNL)

PNL01 Standard Operating Procedure for Laboratory Equipment and Supplies
Standard Operating Procedure for Preparation of Reagents Used in the Standardized
PNL02 PFGE Protocols
PNL03 Standard Operating Procedure for PFGE of Campylobacter
PNL04 Standard Operating Procedure for PFGE of Listeria monocytogenes
PNL05 Standard Operating Procedure for PFGE of E. coli O157:H7, Shigella , and Salmonella

PNL06 Standard Operating Procedure for PFGE of Vibrio cholerae and Vibrio parahaemolyticus
Standard Operating Procedure for Image Acquisition and Production of TIFF Files for Data
PNL07 Analysis
PNL08 Standard Operating Procedure for the Generation of Records and Reports
PNL09 Standard Operating Procedure for Receipt, Shipping, and Storage of Isolates (pending)
Standard Operating Procedure for the Use, Inspection, Cleaning, Maintenance, and
PNL10 Calibration of Equipment
Standard Operating Procedure for Maintenance of Pulsed-Field Gel Electrophoresis
PNL11 Systems
PNL12 Standard Operating Procedure for Maintenance of Dade Microscan Turbidity Meter
PNL13 Standard Operating Procedure for Waterbath Maintenance and Cleaning
Standard Operating Procedure for the Use, Storage, and Disposal of Chemicals and
PNL14 Reagents
PNL15 Standard Operating Procedure for Storage and Use of Expired Reagents
Standard Operating Procedure for Evaluation and Correction of Failure of Any Component
PNL16 of Standardized PFGE Protocol
PNL17 Standard Operating Procedure for Laboratory Training
PNL18 Standard Operating Procedure for PFGE of Yersinia pestis
Laboratory Standard Operating Procedure for PulseNet MLVA of Shiga Toxin-Producing
Escherichia coli O157 (STEC O157) and Salmonella Enterica Serotypes Typhimurium and
PNL19 Enteritidis – Beckman Coulter CEQ 8000/8800/GeXP Platform
 Standard Operating Procedure for adding Thiourea to 0.5X TBE Buffer for Strains of E.
coli O157:H7, Salmonella , Vibrio and other Species or Genera that are "Untypeable" by
PNL20 PFGE

Laboratory Standard Operating Procedure for PulseNet MLVA of Shiga Toxin-Producing

Escherichia coli O157 (STEC O157) and Salmonella Enterica Serotypes Typhimurium
PNL23 and Enteritidis-Applied Biosystems Genetic Analyzer 3130 Platform
PNL25 Standard Operating Procedure for PFGE of Clostridium botulinum
Laboratory Standard Operating Procedure for PulseNet MLVA of Shiga Toxin-Producing
Escherichia coli O157 (STEC O157) and Salmonella Enterica Serotypes Typhimurium
PNL28 and Enteritidis – Applied Biosystems Genetic Analyzer 3500 Platform

PNL31 Laboratory Standard Operating Procedure for PulseNet PFGE of Cronobacter Species

Standard Operating Procedures for the PulseNet Databases (PND)

PND01 Standard Operating Procedure for Computer Equipment and Supplies
PND02 Standard Operating Procedure for Image Analysis Using BioNumerics Software
PND03 Standard Operating Procedure for PulseNet Listserv Access and Use
PND04 Standard Operating Procedure for PFGE Gel Analysis
PND05 Standard Operating Procedure for BioNumerics Administrative Duties (pending)
Standard Operating Procedure for Obtaining and Updating Organism Serotype Codes
PND06 (pending)
Standard Operating Procedure for Updating and Maintaining Gel Analysis Software and
PND07 PulseNet Scripts (pending)
PND08 Standard Operating Procedure for Setup and Use of SecurID Key Fob
PND09 Standard Operating Procedure for Authenticating and Connecting to the Server
PND10 Standard Operating Procedure for Database Training
Standard Operating Procedure for Beta Testing BioNumerics Scripts for New PulseNet
PND11 Databases (pending)
PND12 Standard Operating Procedure for Naming PulseNet Outbreaks and Clusters
PND13 Standard Operating Procedure for Naming PulseNet PFGE Patterns (pending)
PulseNet Standard Operating Procedure for Analysis of MLVA Data of Shiga Toxin-
Producing Escherichia coli O157 (STEC O157) and Salmonella Enterica Serotypes
Typhimurium and Enteritidis in BioNumerics-Beckman Coulter CEQ 8000/8800/GeXP
PND14 Data
PulseNet Standard Operating Procedure for Analysis of MLVA Data of Shiga Toxin-
Producing Escherichia coli O157 (STEC O157) and Salmonella Enterica Serotpyes
Typhimurium and Enteritidis in BioNumerics-Applied Biosystems Genetic Analyzer
PND16 3130/3500 Data

Standard Operating Procedures for the PulseNet QA/QC Program (PNQ)

PNQ01 Standard Operating Procedure for TIFF Quality Grading Guidelines
PNQ02 Standard Operating Procedure for Certification of PulseNet USA Personnel
PNQ03 Standard Operating Procedure for Evaluation of Certification Files
PNQ04 Standard Operating Procedure for the PulseNet USA Proficiency Testing Program
Standard Operating Procedure for MLVA Certification of PulseNet Personnel for the
PNQ05 Beckman Coulter CEQ 8000 Platform
Standard Operating Procedure for MLVA Certification of PulseNet Personnel for the
PNQ06 Applied Biosystems Genetic Analyzer 3130XL Platform
 STANDARD OPERATING PROCEDURE FOR WRITING STANDARD OPERATING CODE: PNG01
Effective Date:
PROCEDURES 05 09 05

1. PURPOSE: To describe the guidelines for the creation, review, and approval of procedures regulating PFGE
activities used by PulseNet.

2. SCOPE: This procedure applies to all PulseNet procedures written in relation to the standardized PFGE
protocols and analysis.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 PFGE: Pulsed-field Gel Electrophoresis
3.3 Effective Date: the day the procedure will be in operation and from which any revisions, corrections, and
distribution times may be calculated.
3.4 QA/QC: Quality Assurance/Quality Control

4. RESPONSIBILITIES:

4.1 The correct format for an SOP can be found in Appendix PNG01-1.
4.2 Assign a concise title to the procedure that gives a general idea of the procedure’s contents.
4.3 Assign a code to the procedure, containing five digits, assigned as follows:
4.3.1 Acronym PN, which refers to PulseNet, and either a G for General SOPs, L for Laboratory SOPs, D for
Database SOPs, or Q for QA/QC SOPs.
4.3.2 Two digit consecutive number of the procedure. For example: The “STANDARD OPERATING
PROCEDURE FOR WRITING STANDARD OPERATING PROCEDURES” is the first General procedure
for PulseNet, so it has the code “PNG01.”
4.4 SOPs should be detailed enough to provide meaningful direction to personnel who conduct routine laboratory
activities.
4.5 SOPs will be reviewed every two years after the effective date, although reviews may be done sooner if
significant changes in the procedure occur.
4.5.1 SOPs will be reviewed by the original author or laboratory supervisor.
4.5.1.1 If changes in the procedure will represent a new standard protocol, the SOP should be reviewed and
the revision approved.
4.5.1.2 If an exception to an SOP is to be made for an individual study, that exception must be authorized in
writing by the laboratory chief or supervisor.
4.5.2 Any changes made must be documented in section 8 of the procedure Amendments.
4.5.3 Author and laboratory supervisor will sign and date in the appropriate areas.
4.5.4 Authorization will be signed and dated by the laboratory chief or director.
4.6 The page number will be written in the lower right corner of each page.
4.7 All personnel following the SOP must read, sign, and date the corresponding Reading Control Sheet (Appendix
PNG01-2).
4.8 Personnel performing activities must have access to the related procedure.
4.8.1 The master of an SOP must be authorized, signed, and dated by the laboratory chief or supervisor, and an
inventory should be kept of when, where, and to whom copies of the SOP have been distributed.
4.8.2 Electronic and hard copy versions of SOPs must be readily available to personnel.
4.8.3 No unauthorized copies should be made and/or distributed. This will provide better control over the
distribution of SOPs, ensuring that all outdated versions of SOPs are retired.
4.8.4 All previous versions shall be retired when new revisions are distributed.
4.8.4.1 Retired SOPs must have the Out-of-Service date listed in red across the top of each page and be
retained in the laboratory for two years from the retirement date. The master copy must be archived
indefinitely.
4.9 The procedure must include:
4.9.1 PURPOSE: Describes in a clear and simple way the mission of the SOP.
4.9.2 SCOPE: Indicates the persons and/or activities concerned with the procedure.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 5
 STANDARD OPERATING PROCEDURE FOR WRITING STANDARD OPERATING CODE: PNG01
Effective Date:
PROCEDURES 05 09 05

4.9.3 DEFINITIONS/TERMS: Provides meanings of abbreviations or important words contained in the

procedure. Appropriate terms and their definitions are listed in Appendix PNG01-2.
4.9.4 RESPONSIBILITIES: Provides obligations that should be met (i.e., Safety). Will sometimes be combined
with Procedure.
4.9.5 PROCEDURE: Describes each activity in a clear and orderly fashion. Will sometimes be combined with
Responsibilities.
4.9.6 FLOW CHART: Simplifies understanding of the procedure.
4.9.7 BIBLIOGRAPHY: Includes all literature consulted for the procedure.
4.9.8 CONTACTS: Provides contact information for persons mentioned within the SOP.
4.9.9 AMENDMENTS: Tracks all changes, updates, and corrections to a procedure.

5. PROCEDURE:

6. FLOW CHART:

7. BIBLIOGRAPHY:

Weinberg, Sandy. GOOD LABORATORY PRACTICE REGULATIONS. Second edition. Marcel Dekker,
Inc. USA (1995).

8. CONTACTS:

9. AMENDMENTS:

Appendix PNG01-1
HEADER SIMILAR TO ABOVE

1. PURPOSE:

2. SCOPE:

3. DEFINITIONS/TERMS:

4. RESPONSIBILITIES:

5. PROCEDURE:

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:

9. AMENDMENTS:

FOOTER SIMILAR TO BELOW

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 5
 STANDARD OPERATING PROCEDURE FOR WRITING STANDARD OPERATING CODE: PNG01
Effective Date:
PROCEDURES 05 09 05

Appendix PNG01-2

Terms and Definitions:

1. SOP: Standard Operating Procedure

2. PFGE: Pulsed-field Gel Electrophoresis
3. TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
4. CDC: Centers for Disease Control and Prevention
5. BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
6. EIS: Epidemic Intelligence Service. EIS Officers are epidemiologists who are given two-year
appointments to help in the investigation of foodborne outbreaks
7. PulseNet liaison: Contact for Foodborne and Diarrheal Diseases Branch epidemiologists and
PulseNet Database Administration Team
8. MSDS: Material Safety Data Sheets
9. PPE: Personal Protective Equipment (goggles, gloves, lab coat, etc.)
10. PFGE inbox: An e-mail account that is maintained and checked by all database managers at CDC.
The address is: [email protected]
11. SecureID key fob: Token that displays a six-digit passcode. When used in combination with a four-
digit pin number, allows access through the CDC firewall
12. Effective date: The day the procedure will be in operation and from which any revisions,
corrections, and distribution times may be calculated
13. N/A: Not Applicable
14. QA/QC: Quality Assurance/Quality Control
15. NCID: National Center for Infectious Diseases
16. BMD: Bacterial and Mycotic Diseases
17. FOIA: Freedom of Information Act
18. OCOO: Office of the Chief Operating Officer
19. PulseNet laboratory: A laboratory that performs PFGE using the approved CDC protocols and
receives support funding via the Epidemiology and Laboratory Capacity (ELC) or Emerging
Infections Program (EIP) federal grants
20. PulseNet Certification Program: Program that tests a individual’s ability to perform PFGE, perform
analysis, create a bundle, and upload a specific organism to the National Database using the PulseNet
standardized protocols.
21. PulseNet Proficiency Testing Program: Program that regularly tests the ability of an individual or
laboratory to perform PFGE and pattern analysis
22. Support zone: A group of state and local health departments that can utilize laboratory services
provided by their Area Laboratory
23. Area Laboratory: Laboratory, designated by CDC, which has agreed to assume responsibility for
additional PulseNet duties for laboratories within their support zone. The current Area Laboratories
include MA, MN, WA, TX, VA, UT, MI and CDC
24. Cluster: A group of isolates with the same serotype determined to possess indistinguishable PFGE
DNA patterns using one or more enzyme restrictions
25. DNA: Deoxyribonucleic acid

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 5
 STANDARD OPERATING PROCEDURE FOR WRITING STANDARD OPERATING CODE: PNG01
Effective Date:
PROCEDURES 05 09 05

26. PulseNet laboratory protocols: Protocols or standard operating procedures followed by all
laboratories participating in the PulseNet program in order to submit DNA fingerprint patterns for
inclusion in the National Database
27. Troubleshoot: To investigate a problem and come up with solutions
28. Surge capacity: Ability to provide additional testing when workload exceeds existing capacity
29. Bundle file: A file with a .bdl extension that is produced in BioNumerics and contains the analysis
of at least one lane of a gel image and may include specified isolate demographic information
30. Foodborne disease epidemiologist: An individual who studies and/or investigates the transmission
and control of foodborne diseases
31. Outbreak: A rise in disease rates as a result of an association or exposure to a specific vehicle
32. Proficiency testing: An annual assessment of the quality of the work being performed in PulseNet
participating laboratories. For each pathogen, proficiency testing includes two parts – a “TIFF sent by
CDC” and a “TIFF generated by the participating laboratory (i.e., in-house TIFF)”
33. Proficiency testing packet: A package sent to the PulseNet participant with the results of his/her PT
evaluation. Includes a hard copy of the cover letter, report, in-house TIFF, and submission e-mail
34. TIFF sent by CDC: This is a part of the proficiency testing program where all laboratories analyze
the same TIFF sent to them by CDC
35. TIFF generated by the participating laboratory, also called the “in-house TIFF”: A part of the
proficiency testing program where laboratories run a gel and produce a TIFF of the gel that contains
the Salmonella Braenderup H9812 standards and the proficiency testing strain restricted with the
primary and secondary enzymes. The TIFF is analyzed and submitted to the on-line database
36. Proficiency Testing survey: An annual survey consists of two rounds of testing, a fall round and a
spring round
37. Comparison list: A list of analyzed lanes from comparison TIFFs that is saved in BioNumerics and
used to compare to the analysis of the submitted TIFF by the certification file evaluator and the
analysis of the submitter in the certification bundle file
38. Gel certified: Formerly “TIFF certified.” An individual or laboratory that is certified in laboratory
methods for PFGE and image acquisition
39. Analysis certified: An individual who is certified in BioNumerics gel analysis
40. Certification files: TIFF and/or bundle files submitted by PulseNet participants for certification
evaluation
41. Certification file evaluator: An individual who evaluates certification files
42. TIFF quality: The grading of the appearance and ease of analysis of a TIFF according to the
PulseNet TIFF Grading Guidelines. This is a main component of the evaluation of a TIFF submitted
for certification
43. Gel analysis assessment: The grading of the whole analysis of a TIFF, including gel and lane
definition, normalization, and band marking, according to the PulseNet Gel Analysis Guidelines. This
is a main component of the evaluation of a bundle file submitted for certification
44. Certification file reviewer: An individual who reviews and signs off on the certification reports
submitted by the certification file evaluator
45. Comparison TIFFs: One or more TIFFs run by CDC for a specific pathogen for use in comparing
PFGE patterns and band resolution against submitted certification TIFFs. Comparison TIFFs can also
be a group of certification TIFFs submitted by several laboratories to monitor PFGE patterns and
band resolution over several submitting laboratories. The latter is most easily accomplished through a
saved list in BioNumerics
46. APHL: Association of Public Health Laboratories
47. Host lab: Term used to describe laboratory hosting a training course
48. OHS: Office of Health and Safety
VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 5
 STANDARD OPERATING PROCEDURE FOR WRITING STANDARD OPERATING CODE: PNG01
Effective Date:
PROCEDURES 05 09 05

49. Training Personnel: Term used to describe PulseNet participants who have been approved by CDC
to train other PulseNet participants

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 5
 STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

1. PURPOSE: To describe the responsibilities and procedure associated with processing in and out of PulseNet.

2. SCOPE: This procedure applies to all PulseNet participating laboratories and personnel. If you are a member of
a laboratory that would like to become a PulseNet participant, please refer to PNG05, Responsibilities for a
PulseNet Laboratory and Standard Operating Procedure for Becoming a PulseNet Laboratory.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 PFGE: Pulsed-field Gel Electrophoresis
3.3 QA/QC: Quality Assurance/Quality Control
3.4 APHL: Association of Public Health Laboratories
3.5 CDC: Centers for Disease Control and Prevention
3.6 PulseNet Listserv: A closed, electronic web conference used for communication among PulseNet participants.
The PulseNet Listserv is open to all laboratory staff at PulseNet participating laboratories. Epidemiologists
working in collaboration with these laboratories, when approved by the PulseNet laboratory contact, U.S. food
regulatory staff, and PulseNet International representatives may also have access. The PulseNet Listserv is not
open to university or private industry personnel unless their inclusion is deemed to be in the interest of public
health. The current program being used is WebBoard.
3.7 SecurID key fob: A secure authentication device used to allow access to resources on the CDC network.
3.8 PulseNet News: The newsletter distributed by PulseNet on a tri-annual basis.

4. RESPONSIBILITIES:
4.1 PulseNet laboratories are expected to notify the PulseNet Database Team and/or APHL (see Contacts section 8
below) when new laboratory staff begin performing PulseNet-related duties and/or when existing staff will no
longer be performing PulseNet-related duties.
4.2 Personnel listed within section 5.1.3.1, “The Distribution List” are responsible for updating the contact
information and any associated documents as follows (see Appendix PNG02-1 for Steps to be Taken at CDC):
4.2.1 PulseNet Database Unit Chief sends the “New Contact Information Sheet” (appendix PNG02-3) to new
PulseNet laboratory staff to complete and return to [email protected]
4.2.2 PulseNet Database Team
4.2.2.1 The PulseNet Database Team keeps the PFGE contacts list up-to-date, including but not
limited to:
4.2.2.1.1 Adding new participants and their contact information
4.2.2.1.2 Updating existing contacts with new information and tracking any changes within the
information box with the date the change was made
4.2.2.1.3 Removing any contacts that no longer perform PulseNet-related duties
4.2.2.2 The PulseNet Database Team also sends an email (see appendix PNG02-2 for template) to a
participant who is either departing or will no longer be performing PulseNet-related duties to make
sure to return their SecurID key fob to CDC and provide CDC and/or APHL with any other pertinent
information that might be available (i.e. plans to hire a new person to take over PulseNet-related
duties). The email will be cc’ed to the main contact of the participant’s Area Laboratory.
4.2.3 IT Support
4.2.3.1 PulseNet Listserv Access
4.2.3.1.1 Adding new participants as their access is approved
4.2.3.1.2 Removing participants who no longer perform PulseNet-related duties
4.2.3.2 Access to National Database(s)
4.2.3.2.1 As participants are certified, assigning SecurID key fobs and access to national database(s)
4.2.3.2.2 Collecting SecurID key fobs and removing access to national database(s) once notified that
participant is no longer performing PulseNet-related duties
4.2.3.3 Information Update
4.2.3.3.1 Emails participants with access to the National Database(s) to verify contact information on
an annual basis
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 10
 STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

4.2.4 QA/QC
4.2.4.1 Updating all certification and proficiency testing documents as participants are granted access
to the national database(s) and as participants are no longer performing PulseNet-related duties
4.2.5 Newsletter
4.2.5.1 Updating newsletter distribution documents with any new or changed contact information and
removing participants who no longer perform PulseNet-related duties
4.2.5.2 Including welcome or farewell announcement in the next issue of the newsletter

5. PROCEDURE:
5.1. New PulseNet personnel
5.1.1. When a laboratory staff member begins performing PulseNet-related duties, the PulseNet Database
Team and APHL must be notified.
5.1.2. The laboratory must contact the PulseNet Database Team and/or APHL with the new person’s contact
information including name, title, position within the lab, mailing address, email address and phone
number. Other information such as fax number and time spent performing PulseNet-related duties
would also be useful.
5.1.3. Once contact information is received, the CDC or APHL person who received this information is
responsible for sending it to everyone on “The Distribution List.”
5.1.3.1. The Distribution List:
PulseNet Database Unit: PFGE Inbox [email protected]
PulseNet Database Unit Chief: Kelley Hise [email protected]
PulseNet Methods Development and Reference Unit Chief: Efrain Ribot [email protected]
Listeria Identification and Subtyping Unit Chief: Lewis Graves [email protected]
IT Support: Brenda Brown [email protected]
QA/QC: Jennifer Kincaid [email protected] and Deborah Sheehan [email protected]
Newsletter: Nehal Patel [email protected]
APHL: Sharon Rolando [email protected]
PulseNet International: Ahmed ElSedawy [email protected] (only include on distribution
list if information is regarding a PulseNet International participant)
5.1.4. Information is added to the PFGE contact list
5.1.5. Once the participant is analysis-certified, access to the national database(s) may be granted and
participant will receive a SecurID key fob (please refer to SOPs PNQ02 and PND08 for information on
PulseNet certification and SecurID key fobs)
5.1.6. PulseNet Listserv access may be requested (please refer to SOP PND03 for information on requesting
access and use of the PulseNet Listserv)
5.1.7. Welcome announcement will be printed in PulseNet News and contact information will be added to the
newsletter distribution list
5.2. Change in contact Information
5.2.1. If contact information should change, participants must notify the PulseNet Database Team and/or
APHL.
5.2.2. CDC will email participants who have access to the National Database(s) to verify contact information
on an annual basis.
5.2.3. Once updated contact information is received, the CDC or APHL person who received this information
is responsible for sending it to everyone on “The Distribution List” (see section 5.1.3.1 above) so that
the appropriate people are notified and information may be updated promptly.
5.3. Participant departs from a PulseNet Laboratory (this includes changing positions or leaving the laboratory)
5.3.1. When a laboratory staff member is no longer performing PulseNet-related duties
5.3.1.1. The PulseNet Database Team and APHL must be notified
5.3.1.1.1. Once the PulseNet Database Team and/or APHL has been notified that a participant has
left their current position and is no longer performing PulseNet-related duties, the CDC
or APHL person who received this information is responsible for sending it to everyone
on “The Distribution List” (see section 5.1.3.1 above).
VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 10
STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

5.3.1.2. If the laboratory staff member was the main PulseNet contact, contact information for the
person who will serve as the replacement/interim contact person should be provided.
5.3.1.3. If the participant was analysis-certified, access to the national database(s) will be removed and
the SecurID key fob must be returned to CDC.
5.3.1.3.1. Please return key fob to:
Centers for Disease Control
Attn: Mike Korth
1600 Clifton Rd. NE
MS-C03
Atlanta, GA 30333
(404) 639-3334
5.3.1.4. Access to the PulseNet Listserv will be removed
5.3.1.5. Information will be removed from the PFGE contact list
5.3.1.6. Farewell and/or change in duties announcement will be printed in PulseNet News and contact
information will be removed from the newsletter distribution list
5.3.1.7. If the laboratory staff member was promoted or reassigned to a different position within their
current laboratory and will no longer be performing routine PulseNet duties, but will serve as a
PulseNet backup, access to the PulseNet Listserv and the national database(s) (if participant
was analysis-certified) may be retained; therefore the staff member should keep their assigned
SecurID key fob. They will not be removed from the PFGE contact list, but their information
should be updated and a note must be written in the information box to indicate this
participant’s new role.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 10
 STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

6. FLOW CHARTS:

New person performing PulseNet

related duties

Laboratory must provide contact

information (name, title, position
within lab, mailing address, email
address, and phone number) to
CDC and/or APHL

Person at CDC or APHL who receives

new contact information must forward
to “The Distribution List” so that all
records may be updated as
necessary. PulseNet Database Unit
Chief sends “New Contact Information
Sheet” to new personnel to complete
and return to [email protected].

New information is added to

PFGE contact list, welcome
published in newsletter, added
to newsletter distribution list,
WebBoard access requested,
QA/QC documents updated,
access to national database(s)
may be granted

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 10
STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

Contact information
changes

Laboratory must notify CDC

and/or APHL

Person at CDC or APHL who

receives new contact
information must forward to
”The Distribution List” so that
all records may be updated
as necessary

Information is updated in the

PFGE contact list, newsletter
distribution list, IT and QA/QC
documents

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 10
 STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

Personnel no longer performing

PulseNet-related duties
(includes change of position or
departing the laboratory)

Laboratory must notify CDC

and/or APHL

Person at CDC or APHL who

receives this information must
forward to the distribution list
so that all records may be
updated as necessary

Participant changes Participant changes positions

positions but will serve and will no longer perform
as PulseNet backup PulseNet duties or participant
departs laboratory

Information is updated within

PFGE contact list to note change Information is removed from
in role, access to WebBoard and PFGE contact list, farewell
national database(s) may be published in newsletter, removed
maintained and SecurID key fob from newsletter distribution list,
kept. Other items such as WebBoard access removed,
newsletter and QA/QC documents QA/QC documents updated,
to be updated as necessary access to national database(s)
depending on new role. removed and SecurID key fob
returned to CDC

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 10
 STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

7. BIBLIOGRAPHY:

8. CONTACTS:

8.1. APHL
Sharon Rolando
Senior Manager for Food Safety
8515 Georgia Avenue
Suite 700
Silver Spring, MD 20910
[email protected]
(240) 485-2777

8.2. CDC PulseNet Database Team

[email protected]
(404) 639-4558

9. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 10
STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

Appendix PNG02-1

Steps to be Taken at CDC

CDC PulseNet personnel are responsible for sending information regarding new PulseNet
participants, updated contact information, and participants no longer performing PulseNet-related
duties to “The Distribution List” (listed in PFGE contacts as “1. The Distribution List,” refer to
section 5.1.3.1 of SOP PNG02 for more information). This appendix may serve as a “what happens
next” document for internal use. Names of the person(s) responsible for each task may be inserted in
the blanks for reference. The following may take place simultaneously. Please refer to SOP PNG02
for more specific information on responsibilities and procedure.

ƒ PulseNet Database Team Member moves email to “Contacts to Update” folder within the
PFGE inbox.
__________________________

√ Personnel responsible for checking the “Contacts to Update” folder will add, update, or
remove information within the PFGE contacts as necessary.
__________________________

ƒ If the PulseNet participant has notified CDC that they will no longer be performing PulseNet-
related duties, the PulseNet Database Unit Chief sends them an email (see appendix PNG02-2
for template) to make sure they return their SecurID key fob (if analysis-certified) and to let them
know that access to WebBoard and the National Database(s) will be removed. The email will be
cc’ed to the main contact of the participant’s Area Laboratory. If there is a new PulseNet
participant, the PulseNet Database Unit Chief will send them the “New Contact Information
Sheet” (appendix PNG02-3) to complete and return to [email protected].
__________________________

ƒ PulseNet IT Support will add, update or remove access information for WebBoard and the
National Database(s) and collect SecurID key fobs as they are returned to CDC. On an annual
basis, will verify contact information for those with access to the National Database(s).
__________________________

ƒ PulseNet QA/QC Manager(s) will add, update or remove certification and proficiency testing
information as necessary.
__________________________

ƒ PulseNet News Editor(s) will update newsletter distribution documents and ensure any
welcomes or farewells are printed in the next issue as appropriate.
__________________________

ƒ PulseNet Methods and Development Reference Unit Chief and the Listeria Identification
and Subtyping Unit Chief will ensure all laboratory personnel and documents are updated as
necessary.
__________________________

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 10
 STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

Appendix PNG02-2

Template: Email to a participant who is either changing positions (no longer performing
PulseNet-related duties) or departing the laboratory and cc the main contact at the
participant’s Area Laboratory

Dear PulseNet Participant,

We are sorry to hear that you will be leaving PulseNet. It has been a pleasure working with you.

If you were analysis-certified, please make sure to return your SecurID key fob to CDC at the
address provided below.

Attn: Michael Korth

Centers for Disease Control
1600 Clifton Rd, NE
MS-C03
Atlanta, GA 30333
(404) 639-3334

Your access to the PulseNet WebBoard and the national database(s) will be removed as of
mm/dd/yyyy (date leaving).

The main contact for your Area Laboratory has been cc’ed on this email in order to maintain
communication flow.

If you have any questions or additional information (i.e. who will be taking over your PulseNet-
related duties), please let us know.

We wish you the best of luck with all future endeavors.

Thank you for your hard work and dedication,

PulseNet Database Team

VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 10
 STANDARD OPERATING PROCEDURE FOR PROCESSING IN AND OUT OF CODE: PNG02
Effective Date:
PULSENET 03 26 07

Appendix PNG02-3

New contact Information Sheet

Prefix: Mr./Ms./Mrs./Dr.
First Name: ____________________
Last Name: ____________________
Suffix: Jr./Sr./PhD/MS/MPH/other __________
Position Title: ____________________
Hire Date: ___/___/______
% of time spent on PN-related activities: _____%

Public Health Lab: __________________________________

Branch: ________________________________________
Unit: ________________________________________

Business: ( ) _____-________ Ext. ______

Fax: ( ) _____-________
E-mail1: ______________________________
E-mail2: ______________________________

Address: ______________________________
______________________________
City: __________________ State: _____ Country: _____________ Zip: __________

Would you like to receive the PulseNet Newsletter? Yes ____ No ____

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 10
 STANDARD OPERATING PROCEDURE FOR HANDLING INQUIRIES FROM CODE: PNG03
Effective Date:
MEMBERS OF THE MEDIA 5 9 05

1. PURPOSE: To describe guidelines to handle inquiries from members of the media.

2. SCOPE: This procedure applies to all PulseNet, NCID, CDC media-related inquiries.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 NCID: National Center for Infectious Diseases
3.3 CDC: Centers for Disease Control and Prevention

4. RESPONSIBILITIES:

4.1 If approached by the media in the office, at home, or on the road, NCID staff should notify NCID media
relations.

5. PROCEDURE:

5.1 Take down the reporter’s name and affiliation and contact NCID media relations.
5.1.1 Contact the NCID Senior Press Officer or Press Officer.
5.2 A press officer will evaluate the request, provide communications guidance, and ensure appropriate
coordination or clearance is achieved.
5.3 Please express to the reporter our willingness to help, but that all requests must come through the press office.
5.3.1 Press officers are available 24 hours a day, seven days a week, 365 days a year.
5.4 In addition, the CDC Division of Media Relations press officers can be reached during work hours at 404-639-
3286, and after hours daily at 404-639-2888. Ask for the on-call press officer.
5.5 For additional information regarding CDC media relations, visit: http://www.cdc.gov/od/oc/media/index.htm

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:
8.1 NCID Senior Press Officer Dave Daigle, W: (404) 639-1143; C: (404) 353-7449; E-mail: [email protected]
8.2 NCID Press Officer, Jennifer Morcone, W: (404) 639-1690; C: (404) 867-7493; E-mail: [email protected]

9. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 1
STANDARD OPERATING PROCEDURE FOR HANDLING INQUIRIES FROM LAWYERS AND CODE: PNG04
Effective Date:
FREEDOM OF INFORMATION ACT (FOIA) REQUESTS
05 09 2005

1. PURPOSE: To describe the steps necessary to address inquiries from lawyers, or Freedom of Information
Act (FOIA) requests.

2. SCOPE: All CDC laboratory personnel must follow this procedure regardless of the source or scope of the
request. All non-CDC labs MUST contact their State Attorney General’s Office for all regulations specific to
their state.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 FOIA: Freedom of Information Act
3.3 BMD: Bacterial and Mycotic Diseases
3.4 OCOO: Office of the Chief Operating Officer
3.5 EIS: Epidemic Intelligence Service: EIS officers are epidemiologists who are given two-year
appointments to help in the investigation of foodborne outbreaks.

4. RESPONSIBILITIES/PROCEDURE:

4.1All requests for FOIA information must come through CDC’s FOIA office.
4.1.1 FOIA office will distribute requests to the branch secretary.
a. The branch secretary will deliver to the appropriate laboratory, OR
b. The branch secretary will deliver to the supervising epidemiologist who determines
who needs to work on it and in what order.
(1) EIS officer
(2) Laboratory
4.1.2 Upon receipt in the laboratory section, administrative support will log the request into the
FOIA tracking logbook.
a. Administrative support will deliver the FOIA request to the laboratory Section Chief
with the tracking form attached.
b. Administrative support will deliver the FOIA request to appropriate laboratory
personnel as indicted on the tracking form.
(1) In the laboratory, FOIAs are received and distributed to appropriate personnel by
the Unit Chief. Once completed, a summary of findings is attached. The Unit Chief
forwards the FOIA request to the next person listed on the routing slip.
(2) Once the last person has seen the FOIA request, it is then returned to administrative
support.
c. After all information has been compiled and FOIA is completed:
(1) Administrative support will return the FOIA request to the laboratory Section Chief
for review and signatures.
(2) Administrative support will perform cost analysis and make copies.
4.1.3 The branch secretary will return the FOIA request to CDC’s FOIA office.
4.2 Freedom of Information Act requests
4.2.1 CDC/FDDB Policy on FOIA Requests
4.2.1.1 Determine if request is reasonable:
a. If yes, provide copies of records after redacting information that may infringe on
another individual’s rights.
b. If no, suggest that requester narrow the scope of the request.
4.2.1.2 Provide the requested information after an ongoing investigation is completed and a
final report is prepared.
4.2.1.3 Provide only existing records; do not create new records.
4.2.1.4 May charge for records (fees for copying, faxing and mailing records).
4.2.2 How to handle requests for food samples
4.2.2.1 No legal basis exists to force compliance with request (CA Supreme Court decision)
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 2
STANDARD OPERATING PROCEDURE FOR HANDLING INQUIRIES FROM LAWYERS AND CODE: PNG04
Effective Date:
FREEDOM OF INFORMATION ACT (FOIA) REQUESTS
05 09 2005

4.2.2.2 General strategy

a. Keep whatever is needed for public health purposes.
b. Give whatever you can spare to a designated laboratory.
c. Direct all sample requests to the person/organization that originally provided
the sample.
d. Send back any unused samples.
4.2.3 How to handle requests for patient isolates
4.2.3.1 No clear directive on sharing isolates.
4.2.3.2 Discretionary, but if you provide isolates to one, you may have to provide to others.
4.2.3.3 CDC/FDDB policy is to wait until an investigation is completed before providing
isolates to outsiders.
4.2.3.4 CDC/FDDB often charges for expenses incurred in providing isolates.
4.2.3.5 If requester provides a public-health-related justification, it is difficult to deny access to
isolates.
4.3 Requests from lawyers
4.3.1 Attorneys may request information from federal establishments under the Freedom of
Information Act.
4.3.1.1 Federal statutes apply:
a. Only to records, not samples or isolates.
b. Only to documents or records that were previously produced (not required to create a
document for a FOIA).
c. Only to those records that do not infringe on a person’s privacy.
4.3.1.2 States may have different requirements
a. Some states have a FOIA equivalent:
(1) Open Records Act
(2) Public Records Act
4.3.2 Refer questions to the laboratory Section Chief or to the attorney’s office in OCOO.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 2
 STANDARD OPERATING PROCEDURE: DUTIES AND RESPONSIBILITIES OF A CODE: PNG05
Effective Date:
PULSENET LABORATORY 09 13 11

1. PURPOSE: To describe the expectations for and responsibilities of a PulseNet participating laboratory.

2. SCOPE: This procedure applies to all PulseNet participating laboratories.

3. DEFINITIONS/TERMS:

3.1 APHL: Association of Public Health Laboratories

3.2 PFGE: Pulsed-field Gel Electrophoresis
3.3 CDC: Centers for Disease Control and Prevention
3.4 PulseNet Laboratory: A laboratory that performs PFGE using the approved CDC protocols and receives
support funding via the Epidemiology and Laboratory Capacity (ELC) or Emerging Infections Program (EIP)
federal grants.
3.5 QA/QC: Quality Assurance/Quality Control
3.6 SOP: Standard Operating Procedure
3.7 PulseNet-related Duties: Duties related to PulseNet work, e.g., preparing PFGE gels, analyzing TIFFs of PFGE
gels, posting to the PulseNet workspace on CDC Team.
3.8 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
3.9 TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
3.10 PulseNet Workspace on CDC Team: A closed, web-based collaboration application used for communication
among PulseNet participants. The PulseNet Workspace on CDC Team is open to all laboratory staff at
PulseNet participating laboratories. Epidemiologists working in collaboration with these laboratories, when
approved by the PulseNet laboratory contact, U.S. food regulatory staff, and PulseNet International
representatives may also have access. The PulseNet Workspace on CDC Team is not open to university or
private industry personnel unless their inclusion is deemed to be in the interest of public health.
(http://team.cdc.gov). For additional information on CDC Team please refer to SOP PND03.
3.11 PulseNet Certification Program: Program that tests an individual’s ability to perform PFGE on a specific
organism using the standard protocols, perform analysis, create bundles, and upload patterns to the National
Database.
3.12 PulseNet Proficiency testing program: Program that regularly tests the ability of an individual or laboratory to
perform PFGE and pattern analysis
3.13 Support zone: A group of state and local health departments that can utilize laboratory services provided by
their Area Laboratory
3.14 PulseNet Area Laboratory: Laboratory, designated by CDC, which has agreed to assume responsibility for
additional PulseNet duties for laboratories within their support zone. The current Area laboratories include
MA, MN, WA, TX, VA, UT, MI and CDC.

4. RESPONSIBILITIES:

4.1 PulseNet laboratories must, at a minimum, perform PFGE on PulseNet-tracked organisms as requested by
CDC or state epidemiologists.
4.1.1 The organisms currently tracked by PulseNet are E. coli O157:H7, Non O157 (STEC), Listeria
monocytogenes, Salmonella, Shigella, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus,
and Clostridium botulinum.
4.1.2 If unable to complete this testing in-house in real time, the laboratory must forward the isolates
immediately to CDC or to the appropriate Area Laboratory for subtyping.
4.2 PulseNet laboratories must adhere to the protocols and requirements of the PulseNet QA/QC manual available
on the PulseNet Workspace on CDC Team.
4.3 Individuals performing PulseNet-related duties must submit certification file(s) and have them reviewed before
being able to submit TIFF images to the PulseNet National Databases (PNQ02).
4.4 Certified laboratories performing PulseNet-related duties must participate in the annual CDC- sponsored
proficiency-testing program (PNQ04).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1of 3
 STANDARD OPERATING PROCEDURE: DUTIES AND RESPONSIBILITIES OF A CODE: PNG05
Effective Date:
PULSENET LABORATORY 09 13 11

4.5 PulseNet laboratories must submit all PFGE patterns and corresponding information to the PulseNet national
database (if certified for pattern upload) or to the PulseNet database team at CDC within 24 hours of the day
results are generated.
4.5.1 Real-time surveillance should be the goal of all PulseNet laboratories and should involve real-time
subtyping of PulseNet tracked organisms that are received by the laboratory.
4.5.2 CDC does not require subtype information of isolates generated by PFGE or another PulseNet subtyping
method, with the same serotype and same PFGE pattern, from the same patient, collected within 6
months of each other to be uploaded to the PulseNet National Databases unless the subtypes of the
isolates are different.
4.5.2.1 If a duplicate isolate (one from the same patient, with the same serotype and PFGE pattern within 6
months) is run by PFGE, please use the best representation of the pattern produced to submit to the
national database.
4.6 PulseNet laboratories will agree to store isolates that have been subtyped by PFGE for as long as space will
allow and for a minimum of 1 year. These isolates will be submitted to CDC when requested for isolate
collections or epidemiologic investigations.
4.7 PulseNet laboratories must work with their epidemiology department on a routine basis for the investigation of
clusters and outbreaks.
4.8 PulseNet staff must sign and submit the “PulseNet CDC Team Non-disclosure Agreement” to receive access to
the PulseNet workspace on CDC Team (see SOP PND03).
4.9 PulseNet laboratories must post information to the PulseNet Workspace on CDC Team on foodborne disease
clusters and outbreaks occurring in their state in a timely manner and must respond to postings within 48 hours
(PND03).
4.10 PulseNet laboratories must send at least one representative to the Annual PulseNet Update Meeting.
4.11 PulseNet laboratories must send at least one representative to Regional PulseNet Meetings hosted by their
assigned Area Laboratory.
4.12 PulseNet laboratories are expected to notify APHL, their Area Laboratory, and CDC when new laboratory
staff begins performing PulseNet-related duties and/or when existing laboratory staff will no longer be
performing PulseNet-related duties (see SOP PNG02).
4.13 PulseNet laboratories are expected to write articles for the PulseNet News newsletter when requested by
APHL and/or CDC.
4.14 PulseNet Laboratories will participate in national and regional conference calls organized by APHL, their Area
Laboratory, or CDC.

5. PROCEDURES:

5.1 Labs that are interested in becoming a PulseNet participant must contact the PulseNet Program Manager at
APHL and the PulseNet Database Team at CDC (see SOP PNG08 for information on becoming a PulseNet
Laboratory).
5.2 Prior to being considered a PulseNet laboratory, the laboratory must acquire the necessary equipment, reagents
and supplies, communication tools, and software (PNL01, PNL02, PND01).
5.3 PulseNet participating laboratories must complete a PulseNet-sponsored training (PNL17, PND10), subscribe
to the PulseNet Workspace on CDC Team (PND03), and request the Salmonella ser. Braenderup H9812
standard strain, the latest version of the PulseNet MasterScripts for the PulseNet customized version of
BioNumerics from the PulseNet Database Team, and a copy of the PulseNet QA/QC Manual. All laboratories
are expected to maintain knowledge of the current laboratory methods and software configurations utilized by
the network.
5.4 PulseNet laboratories must perform PFGE digested with two enzymes on all E. coli and Listeria
monocytogenes isolates immediately after receipt in their laboratory.
5.5 When PulseNet participating labs submit data to the National Database(s) (either directly or to the PulseNet
Database Team), as much information as possible should be included. At a minimum, the required information
includes (using pick lists whenever applicable):
5.5.1 City and/or County (if known)
5.5.2 State
VERSION: REPLACED BY: AUTHORIZED BY:

Page 2of 3
STANDARD OPERATING PROCEDURE: DUTIES AND RESPONSIBILITIES OF A CODE: PNG05
Effective Date:
PULSENET LABORATORY 09 13 11

5.5.3 Species/Serotype (even if this is undetermined, pending, untypable)

5.5.4 Source Type (even if unknown)
5.5.5 Source Site (even if unknown)
5.5.6 Other State Isolate Number (when applicable)
5.5.7 Isolation Date
5.5.8 Received Date
5.5.9 Age and Sex

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:

8.1 APHL:
Kristy Kubota MPH
Association. of Public Health Laboratories
Phone (240) 485-2720
Fax (240) 485-2700
Kristy. [email protected]

8.2 Centers for Disease Control and Prevention:

Kelley Hise MPH
Phone: (404) 639-0704
PulseNet: (404) 639-4558
Fax: (404) 639-3333
[email protected]

9. AMENDMENTS:
9.1 2011-09-13 Section 4.11 was added: PulseNet laboratories must send at least one representative to
Regional PulseNet Meetings hosted by their assigned Area Laboratory.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3of 3
 STANDARD OPERATING PROCEDURE FOR CODE: PNG06
Effective Date:
PULSENET AREA LABORATORIES 5 9 05

1. PURPOSE: To describe the responsibilities and duties of a PulseNet Area Laboratory.

2. SCOPE: This procedure applies to all PulseNet Area Laboratories.

3. DEFINITIONS/TERMS:

3.1 PFGE: Pulsed-field Gel Electrophoresis

3.2 CDC: Centers for Disease Control and Prevention
3.3 SOP: Standard Operating Procedure
3.4 Area laboratory: Laboratory, designated by CDC, which has agreed to assume responsibility for additional
PulseNet duties for laboratories within their support zone. The current Area Laboratories include MA, MN,
WA, TX, VA, UT, MI, and CDC
3.5 Support zone: A group of state and local health departments that is served by a particular Area laboratory
3.6 BioNumerics: Gel analysis software used by PulseNet developed by Applied Maths, Belgium
3.7 PulseNet Listserv: Secure, closed unmoderated Listserv used by registered PulseNet participants to post
possible clusters or outbreaks, PulseNet-related questions, technical information, and responses to postings
3.8 Cluster: A group of isolates with the same serotype with indistinguishable PFGE DNA patterns by one or more
restriction enzyme
3.9 DNA: Deoxyribonucleic acid
3.10 PulseNet Standardized PFGE 24 hour Protocols: Protocols or standard operating procedures followed by all
laboratories participating in the PulseNet program in order to submit DNA fingerprint patterns for inclusion in
the National Database
3.11 Troubleshoot: To investigate a problem and come up with solutions
3.12 Surge capacity: Ability to provide additional testing when workload exceeds existing capacity
3.13 Bundle file: A file with a .bdl extension that is produced in BioNumerics and contains the analysis of at least
one lane of a gel image and may include specified isolate demographic information
3.14 Foodborne disease epidemiologist: A trained individual who studies and/or investigates the transmission and
control of foodborne diseases
3.15 Outbreak: A cluster of cases of infections with a common epidemiological exposure, e.g. to a specific food
product.
3.16 QA/QC: Quality Assurance and Quality Control

4. RESPONSIBILITIES/ PROCEDURES:

4.1 PulseNet Area Laboratories must fulfill the responsibilities of all PulseNet laboratories (PNG05).
4.2 Train laboratory personnel in their support zone to perform PFGE, image analysis, and data analysis using
BioNumerics software when requested.
4.2.1 Train laboratory personnel on all aspects of the PulseNet Standardized PFGE protocols and the safety
requirements associated with performing the testing. Upon completion of training, the trainees should be
competent to perform all steps of the protocol, including preparing plugs, performing cell lysis, digesting
DNA with a restriction endonuclease, preparing and loading an agarose gel, and preparing all required
reagents.
4.2.2 Train laboratory personnel on image acquisition and the use of BioNumerics software (version 3.5 or
greater). Upon completion of training, laboratory personnel will be competent to visually evaluate gel
and fingerprint quality using the PulseNet TIFF Quality Grading Guidelines released in May 2004,
recognize reasons for poor banding or image quality (i.e. incomplete restriction, ghost bands, sub par
resolution, etc.), provide technical suggestions for quality improvements, and electronically save
acceptable gel images as TIFF files. Laboratory personnel will be competent to use BioNumerics
software to define a gel strip lane, normalize a gel, correctly assign DNA fingerprint band positions
according to the PulseNet Gel Analysis Guidelines (PND04), compare PFGE DNA fingerprint patterns to
a locally established database, and generate a bundle file for submission of images to the PulseNet
mailbox ([email protected]).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 3
 STANDARD OPERATING PROCEDURE FOR CODE: PNG06
Effective Date:
PULSENET AREA LABORATORIES 5 9 05

4.2.3 Once a lab is certified for analysis and receives a SecurID fob, they should be trained on how to compare
patterns in their local database(s) to patterns on the national database. Advanced BioNumerics training is
encouraged for all personnel.
4.2.4 Train laboratory personnel how to access the PulseNet Listserv, respond to postings, post a new cluster
(PND03), download a bundle from the Listserv, and compare the downloaded pattern to a local database
to identify possible matching fingerprint patterns.
4.3 Provide PFGE troubleshooting assistance to states in their support zone when requested.
4.3.1 Work with laboratory personnel to critically troubleshoot and identify possible solutions to technical
problems with gel quality, image analysis or fingerprint pattern analysis using BioNumerics software.
4.3.2 Provide laboratory personnel with appropriate contact person(s) at the CDC to address problems the area
laboratory is not able to resolve.
4.4 Provide PulseNet surge capacity for laboratories in an area laboratory support zone when requested.
4.4.1 In instances where a laboratory cannot perform PFGE on all mandatory isolates (E. coli 0157:H7,
Listeria monocytogenes, and designated Salmonella serotypes or other organisms involved in an
outbreak), the area laboratory will perform PFGE on these isolates as requested by the support zone
laboratory. Situations requiring area laboratory surge capacity testing support may include outbreaks,
response to enhanced pathogen surveillance, loss of support zone laboratory certification, or staffing
shortages at the support zone laboratory.
4.4.2 Upon completion of PFGE, the area laboratory will upload the PFGE DNA fingerprint patterns to the
appropriate national database.
4.4.3 The area laboratory will email a TIFF and bundle file containing the completed PFGE DNA fingerprint
patterns to the support zone laboratory for inclusion in the state’s local database.
4.5 Coordinate multi-state outbreak investigations in their support zone at CDC’s request.
4.5.1 Provide regular updates to the foodborne disease laboratorians in affected states regarding new isolates
posted to the national database that are indistinguishable from the outbreak pattern.
4.5.2 Communicate with affected states to verify that all isolates associated with an outbreak are being
subtyped by PFGE in real-time, and if not, coordinate transport of isolates to area laboratory for testing.
4.5.3 Provide surge capacity testing for support zone states as necessary.
4.5.4 Update PulseNet Listserv when relevant demographic data, epidemiologic or laboratory information is
available.
4.6 Perform advanced surveillance of non-routine pathogens, including but not limited to Vibrio sp., Clostridium
sp., Campylobacter sp., and rare Salmonella serotypes in their support zone when requested.
4.6.1 Become proficient in the protocols required to perform PFGE on non-routine pathogens.
4.6.1.1 Perform PFGE DNA fingerprinting of non-routine isolates as requested by states within the
support zone.
4.6.1.2 Create and maintain databases for non-routine organisms under surveillance by the PulseNet
program.
4.7 Evaluate new software and laboratory procedures or procedural modifications of existing procedures when
requested by CDC.
4.7.1 Assist with the evaluation and beta-testing of new software scripts and programs or laboratory protocols
from CDC prior to general release.
4.7.2 Provide technical recommendations to the CDC regarding software changes that may improve its use at
the state level.
4.8 Participate in research and development projects independently and with CDC.
4.8.1 Project or grant announcements will be distributed via the PulseNet Listserv and by APHL.
4.8.2 Data should be collected according to the protocol or grant and shared with CDC upon request.
4.8.3 States should be willing to work with CDC and other involved states on joint publications and
presentations, where applicable.
4.9 Provide guidance for program issues and make recommendations for development of the PulseNet network.
4.9.1 Participate on the PulseNet Steering Committee, upon invitation.
4.9.2 Participate in QA/QC meetings, upon request.
4.9.3 Participate in Area Laboratory conference calls.
4.9.4 Review PulseNet documents.
VERSION: REPLACED BY: AUTHORIZED BY:
Page 2 of 3
 STANDARD OPERATING PROCEDURE FOR CODE: PNG06
Effective Date:
PULSENET AREA LABORATORIES 5 9 05

4.9.5 Participate in Annual Update Meeting (speaker, moderator, etc.).

4.10 Host regional and national PulseNet update meetings and training conferences.
4.10.1 Designate a laboratory point of contact to coordinate training conference planning activities.
4.10.2 Provide conference resources as defined in the planning meetings.
4.10.3 Work with designated agencies to provide state and laboratory resources for the conference.
4.10.4 Provide assistance to other Area Lab states or non-Area Lab states that may offer to host the meeting
following location approval by the CDC.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 3
 STANDARD OPERATING PROCEDURE FOR SHARING DATABASE CODE: PNG07
Effective Date:
INFORMATION BETWEEN PULSENET USA AND PULSENET CANADA 12 01 06

1. PURPOSE: To describe the guidelines for the access and use of information within the PulseNet USA and
PulseNet Canada national databases.

2. SCOPE: This SOP applies to PulseNet USA and PulseNet Canada personnel who will have read-only access to
each other’s national databases. Personnel at PulseNet Canada must comply to be granted access to PulseNet
USA’s national databases and personnel at PulseNet USA must comply to be granted access to PulseNet
Canada’s national databases. This SOP is a “living” document, subject to modifications after discussions
between PulseNet USA and PulseNet Canada personnel.

3. DEFINITIONS/TERMS:
3.1 SOP: Standard Operating Procedure
3.2 PFGE: Pulsed-field Gel Electrophoresis
3.3 QA/QC: Quality Assurance/Quality Control
3.4 CDC: Centers for Disease Control and Prevention
3.5 DNA: Deoxyribonucleic acid
3.6 MOU: Memorandum of Understanding
3.7 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
3.8 National database: Database that houses all isolate and image data for a particular organism at the national level
3.9 TIFFs: Tagged Image File Format, a file of a gel image that can be analyzed in BioNumerics
3.10 Bundle File: A file with a .bdl extension that is produced in BioNumerics and contains the analysis of at least
one lane of a gel image and may include specified isolate demographic information
3.11 Cluster: A group of isolates, identified within the past 30 days for Yersinia; 60 days for Salmonella, E. coli,
Shigella, Campylobacter, and Vibrio; and 120 days for Listeria, with the same serotype with indistinguishable
PFGE DNA patterns by one or more restriction enzymes.
3.12 Outbreak: A cluster of cases of infections, identified within the past 30 days for Yersinia; 60 days for
Salmonella, E. coli, Shigella, Campylobacter, and Vibrio; and 120 days for Listeria, with a common
epidemiological exposure, e.g. to a specific food product.
3.13 PulseNet Listserv (CDC PulseNet WebBoard): A closed, moderated, electronic web conference used for
communication among PulseNet participants. The PulseNet Listserv is open to all laboratory staff at PulseNet
participating laboratories. Epidemiologists working in collaboration with these laboratories, when approved by
the PulseNet laboratory contact, U.S. food regulatory staff, and PulseNet International representatives may also
have access. The PulseNet Listserv is not open to university or private industry personnel unless their inclusion
is deemed to be in the interest of public health.
3.14 SecurID key fob: Token that displays a six-digit pass code. When used in combination with a four-digit pin
number, allows access through the CDC firewall.
3.15 VPN: Virtual Private Network
3.16 PulseNet certification program: Program that tests an individual’s ability to perform PFGE on a specific
organism using the standard protocols, perform analysis, create bundles, and upload patterns to the PulseNet
USA national database.
3.17 PulseNet proficiency testing program: Program that regularly tests the ability of an individual or a laboratory to
perform PFGE and pattern analysis.

4. RESPONSIBILITIES AND PROCUDURE:

4.1. Accessing the national databases
4.1.1. Prior to accessing either country’s national database(s), an individual must:
4.1.1.1. Have received approval from their supervisor to request access
4.1.1.2. Be analysis-certified. The individual must submit certification file(s) to CDC and become
analysis-certified according to PulseNet USA’s SOP PNQ02 for the database for which they
are requesting access (e.g. to be granted access to the Salmonella database, one must be
analysis-certified for Salmonella). Within the certification submission email, the individual
must state that he or she is requesting access to the Canadian and/or USA national database.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 3
 STANDARD OPERATING PROCEDURE FOR SHARING DATABASE CODE: PNG07
Effective Date:
INFORMATION BETWEEN PULSENET USA AND PULSENET CANADA 12 01 06

4.1.1.2.1. If an individual in PulseNet Canada is already analysis-certified and needs access to the
USA national database, the individual will receive approval from the PulseNet Canada
Section Head, who will send a request via email to [email protected].
4.1.1.2.2. If an individual in PulseNet USA is analysis-certified and needs access to a PulseNet
Canada national database, the individual will receive approval from the PulseNet USA
Database Team Leader, who will send a request via email to PN_Canada@phac-
aspc.gc.ca.
4.1.2. Individuals accessing the PulseNet USA national database(s) will receive a SecurID keyfob,
instructions on how to set up the PIN (see SOP PND08), database login(s) and password(s).
4.1.2.1. Once analysis-certification is successfully completed, database access will be requested by the
PulseNet USA QA/QC Manager(s)
4.1.2.2. SecurID keyfob, database login(s), and password(s) will be mailed to the individual
4.1.3. Individuals accessing the PulseNet Canada national database(s) will receive a VPN, instructions on
how to set it up, database login(s) and password(s).
4.1.3.1. Once analysis-certification is successfully completed, database access will be requested by the
PulseNet Canada Section Head.
4.1.4. To maintain access to the national database(s), laboratories must successfully complete annual
proficiency testing (for each organism certified) sponsored and conducted by CDC as described in
SOP PNQ04.
4.1.5. Instead of emailing questions about possible matches to ongoing clusters or outbreaks and/or
historical data, each country will be able to access this information directly to perform their own
comparisons on an “as needed basis.”
4.2. Reporting significant findings
4.2.1. If PulseNet USA finds a potential match (or matches) to a cluster or an outbreak within the PulseNet
Canada national database, PulseNet Canada must be notified by email, PN_Canada@phac-
aspc.gc.ca or phone (204) 789-5067. PulseNet Canada is then responsible for deciding if there is
something significant to report and if so, how to proceed (i.e. reporting to epidemiologists, posting to
a listserv, and/or taking any additional action).
4.2.2. If PulseNet Canada finds a potential match (or matches) to a cluster or outbreak within the PulseNet
USA national database, PulseNet USA must be notified by email, [email protected] or phone (404)
639-4558. PulseNet USA is then responsible for deciding if there is something significant to report
and if so, how to proceed (i.e. reporting to epidemiologists, posting to a listserv, and/or taking any
additional action).
4.2.3. If one country wishes to use data found in the other’s database for a project, report, and/or
publication, this must be discussed and approved by both laboratories before work commences.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

7.1 PulseNet USA

Kelley Hise
Database Team Leader
Centers for Disease Control and Prevention
1600 Clifton Road, NE
MS-C03
Atlanta, GA 30333
[email protected]
(404) 639-4558

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 3
 STANDARD OPERATING PROCEDURE FOR SHARING DATABASE CODE: PNG07
Effective Date:
INFORMATION BETWEEN PULSENET USA AND PULSENET CANADA 12 01 06

7.2 PulseNet Canada

Celine Nadon
Research Scientist and Section Head, PulseNet Canada
National Microbiology Laboratory
Public Health Agency of Canada
1015 Arlington Street
Winnipeg MB R3E 3R2
Canada
[email protected], [email protected]
(204) 784-7507

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 3
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
1. PURPOSE: To describe the application process for becoming a PulseNet participating laboratory and to
describe the steps to process this request through CDC and APHL.

2. SCOPE: This procedure applies to CDC, APHL and all laboratories that are interested in becoming PulseNet
participants.

3. DEFINITIONS/TERMS:

3.1. APHL: Association of Public Health Laboratories

3.2. PFGE: Pulsed-field Gel Electrophoresis
3.3. CDC: Centers for Disease Control and Prevention
3.4. USDA: United States Department of Agriculture
3.5. FDA: Food and Drug Administration
3.6. EDLB: Enteric Diseases Laboratory Branch
3.7. QA/QC: Quality Assurance/Quality Control
3.8. SOP: Standard Operating Procedure
3.9. EISO: Epidemic Intelligence Service Officer
3.10. PulseNet-related Duties: Duties related to PulseNet work, e.g., preparing PFGE gels, analyzing TIFFs of PFGE
gels, participation on the PulseNet SharePoint site (duties and responsibilities detailed in SOP PNG05)
3.11. BioNumerics: Gel analysis software used by PulseNet developed by Applied Maths, Belgium
3.12. TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
3.13. PulseNet SharePoint Site: A closed, web-based discussion forum used for communication among PulseNet
and OutBreakNet participants. SharePoint is open to all laboratory staff at PulseNet participating laboratories
and Epidemiologists working in collaboration with these laboratories, U.S. food regulatory staff, and some
PulseNet International representatives may also have access. SharePoint is not open to university or private
industry personnel unless their inclusion is deemed to be in the interest of public health. Throughout this
document, the PulseNet SharePoint site will be referred to as “SharePoint.”
3.14. Certification: Program that tests an individual’s ability to perform PFGE on a specific organism using the
PulseNet standardized protocols, perform analysis, create bundle files, and upload patterns to the national
database.
3.15. Proficiency Testing: Program that annually tests the ability of a certified PulseNet-participating laboratory to
perform PFGE, pattern analysis, and upload results to the proficiency testing database.
3.16. Area Laboratory: Laboratory, designated by CDC and APHL, which has agreed to assume responsibility for
additional PulseNet duties for laboratories within their support region. The current Area Laboratories include
MA, MN, WA, TX, VA, UT, MI and CDC.
3.17. Contacts Database: Access database where all PulseNet contact information, including but not limited to name,
address, phone, fax, email, laboratory information, certification status, mailing lists, and any additional
comments is housed. The database is located
\\cdc\project\CCID_NCZVED_DFBMD_PulseNet\Admin\Contacts.
3.18. PulseNet Steering Committee: A committee chaired by the chief of the EDLB at CDC and comprised of
participants from state, local and agriculture PulseNet USA laboratories, USDA, FDA, APHL, and CDC. The
steering committee meets via conference call several times a year to guide the expansion, improvement, and
evaluation of the PulseNet USA program.

4. RESPONSIBILITIES:
4.1. PulseNet-participating laboratories currently include state, city, county, food regulatory (USDA and FDA),
agriculture and veterinary labs. PulseNet is not open to university or private industry laboratories unless their
inclusion is deemed to be in the interest of public health.
4.2. Prior to being considered a PulseNet laboratory, the lab must acquire the necessary equipment, reagents and
supplies, communication tools, and software (refer to SOPs PNL01, PNL02, PND01, and PND03 for detailed
information).
4.3. Laboratories must agree to follow the responsibilities of a PulseNet-participating laboratory as described in
SOP PND05.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
5. PROCEDURE:
5.1. Laboratories interested in becoming a PulseNet participant must contact CDC (see section 8 for contact
information).
5.1.1. All requests sent to [email protected] will be moved to the folder “Prospects” in the “Participants”
folder within the PFGE inbox.
5.1.2. Once the email has been forwarded to CDC’s PulseNet USA leadership and copied to APHL, the
email should be marked with the forwarder’s initials and flagged with what action was taken (Ex:
KH: fwd to PGS and APHL).
5.2. The PulseNet USA Steering Committee will review and approve or disapprove the laboratory’s request to
become a PulseNet participating laboratory.
5.2.1. If the PulseNet USA Steering Committee does not approve of the laboratory’s participation in
PulseNet, the PulseNet Database Unit Chief must email the laboratory and copy APHL.
5.2.2. If the PulseNet USA Steering Committee approves of the lab’s participation in PulseNet, the
PulseNet Database Unit Chief will determine, in collaboration with APHL, level of participation
expected from the laboratory. For example, some laboratories may only become gel-certified and
may not receive access to the national databases.
5.3. APHL will verify the laboratory’s APHL membership status and determine any costs associated with PulseNet
participation. For example, if a lab is not an APHL member, there are fees associated with PulseNet
MasterScripts, certification, proficiency testing, and training courses held by CDC (see appendix PNG08-1 for
more information).
5.4. If the laboratory is located in a state or facility where there already are PulseNet-participating laboratories, as a
courtesy, the pre-existing PulseNet lab will be notified by APHL. For example, if a state’s agricultural
laboratory would like to participate, the state laboratory must be notified of the agricultural lab’s request to
participate.
5.5. Once CDC and APHL have determined the laboratory’s level of participation, this information is sent via
email to “The Distribution List” (see PNG02 for more information regarding “The Distribution List” and the
In-Processing procedure for new participants) to notify others at CDC and APHL. Contact information (name,
laboratory, address, phone, fax, and email) must also be provided. This information will be entered into the
Contacts Database and the new lab/person will be listed as a “Prospect.”
5.5.1. The Distribution List:
PFGE inbox [email protected]
Kelley Hise [email protected]
Molly Freeman [email protected]
Jennifer Adams [email protected]
Efrain Ribot [email protected]
Deborah Sheehan [email protected]
Eija Trees [email protected]
Kristy Kubota [email protected]

5.6. CDC will email the PulseNet Memorandum of Understanding (appendix PNG08-2) and the Terms of
Reference (appendix PNG08-3) to the prospective laboratory. The laboratory director must agree to abide by
the terms set forth in these documents and return the Memorandum of Understanding signed and dated to
CDC. Address and contact information provided in section 8 below.
5.7. The PulseNet Database Unit Chief will assign a PulseNet LabID. This information is entered in the
spreadsheet “LabIDs.xls” which is saved under
\\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\Admin\PN Participants.
5.7.1. PulseNet LabIDs consist of two to four characters as follows:
5.7.1.1. For state laboratories the LabID is the two letter state postal code
5.7.1.2. For city or county laboratories the LabID is the two letter state postal code and two appropriate
characters designated by the PulseNet Database Unit Chief
5.7.1.3. For government laboratories the PulseNet Database Unit Chief will determine the most
appropriate LabID

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
5.7.1.4. For agricultural laboratories the LabID is the two letter state postal code and “AG” for
agricultural
5.7.1.5. For veterinary laboratories the LabID is the two letter state postal code and “VT” for
veterinary
5.7.1.6. For university laboratories the PulseNet Database Unit Chief will determine the most
appropriate LabID
5.7.1.7. For international laboratories the PulseNet Database Unit Chief will determine the most
appropriate LabID (in some cases the international hubs will assign their own LabIDs).
5.8. The PulseNet Database Unit Chief will send an email welcoming the laboratory to PulseNet and list any
associated costs for non-APHL members. This email will be copied to APHL, the appropriate Area Laboratory
(see appendix PNG08-4 for email template), the person who initiated contact with CDC and/or APHL, and
any associated state laboratories.
5.8.1. Laboratories that must purchase the PulseNet MasterScripts will send payment to APHL as described
in appendix PNG08-1.
5.8.2. The personnel evaluating certifications and proficiency tests will notify APHL by email of
participating laboratories that are not APHL members so they may be billed for this testing according
to PNG08-1.
5.9. The PulseNet Database Unit Chief will send a starter packet to the participating laboratory. The packet will
include:
5.9.1. Welcome letter containing their PulseNet LabID, contact information (including websites) for CDC,
APHL and their Area Laboratory (see appendix PNG08-5 for template)
5.9.2. Responsibilities of a PulseNet Laboratory SOP PNG05
5.9.3. Access and Use of the PulseNet SharePoint site SOP PND03
5.9.4. Certification SOP PNQ02
5.9.5. “Tips for Getting Started with PulseNet” (see appendix PNG08-6)
5.9.6. An excel file listing the PulseNet organisms, their differences, and database information. Document is
updated for the annual EISO training and saved under
\\cdc\project\CCID_NCZVED_DFBMD_PulseNet\Marketing\Presentations\EISO\yyyy\Handouts\Puls
eNet Organisms_differences.xls.
5.10. After six months, if a new PulseNet laboratory does not seem to be participating by submitting certification
sets and/or sending data to CDC, CDC and/or APHL will contact them to check on their status.
5.11. If a PulseNet laboratory can no longer perform PulseNet duties for any reason, the laboratory director must
notify CDC in writing. Depending on the circumstances, arrangements will be made on a lab-to-lab basis.
6. FLOWCHART:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13

Prospective Lab emails PFGE inbox

to inquire about joining PulseNet

Email forwarded to Steering Committee

and APHL and flagged with action taken,
i.e. KH: fwd to PGS and APHL

If the Steering Committee and APHL approve If Steering Committee does not
lab’s participation, they determine the lab’s approve lab’s participation, the
level of participation. For example, some labs PulseNet Database Unit Chief
may only become gel-certified and are not notifies the laboratory as soon as
allowed access to the national databases. this decision is reached.

APHL verifies lab’s APHL membership and determines any potential

costs for the lab (if not APHL member). If the laboratory is located in a
state or facility where there already are PulseNet-participating
laboratories, as a courtesy, the pre-existing PulseNet lab(s) will be
notified by CDC or APHL.

The PulseNet Database Unit Chief emails “The

Distribution List” to notify others at CDC and
APHL of the new lab. Email should include
contact information and lab’s level of
participation. Contact info should be entered
into contacts database. MOU and TOR docs are
sent to the prospective lab.

The PulseNet Database Unit Chief assigns

the new lab a PulseNet LabID, sends a
welcome email to lab and copies APHL and
the appropriate Area Laboratory

After ~6 months, if the new lab has not

shown any signs of PulseNet participation
(i.e. not submitted PFGE patterns or no
certification attempts) CDC or APHL will
contact them to check participation status.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13

7. BIBLIOGRAPHY:

8. CONTACTS:
8.1. CDC PulseNet
Phone (404) 639-4558
Fax (404) 639-3333
[email protected]

9. AMENDMENTS:
9.1. January 2011: Updates made to the entire document to reflect CDC’s reorganization, changes in position
titles, and terms and definitions.
9.2. December 2012: Updates made to change CDC Team to SharePoint, Appendix PNG08-1 updated to
reflect current charges and payment information for laboratories that are not APHL members.
9.3. October 2014: Updates made to enter new PulseNet MOU and ToR documents

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13

APPENDIX PNG08-1
Payment information for federal participants and non-APHL members

PulseNet PFGE and MLVA Certifications and Proficiency Testing

Tier 1 laboratories will be billed $2,000 per year (October 1-September 30)
Includes:
• All PulseNet proficiency tests for organisms in which the laboratory is currently certified
• Up to 3 certifications submitted to PulseNet within the year as defined (these are use or lose, they
do not accrue or roll over to the next year). Additional certifications may be purchased for $500
per organism per laboratorian.

Tier 2 laboratories will be billed $1,000 per year (October 1-September 30)
Includes:
• All PulseNet proficiency tests for organisms in which the laboratory is currently certified.
• Up to 1 certification submitted to PulseNet within the year as defined (these are use or lose, they
do not accrue or roll over to the next year). Additional certifications may be purchased for $500
per organism per laboratorian.

Laboratories are billed for certification and proficiency testing according to a designated Tier classification. Tiers
are determined by CDC and APHL based on several criteria including but not limited to the number of current
employees performing PulseNet-related duties, the number of organisms in which the laboratory is certified (which
also affects the number of proficiency tests), and the past charges incurred by the laboratory for PulseNet QA/QC
related services. The past three years (fiscal years October 1-September 30) worth of certifications and proficiency
tests performed by the laboratory are averaged to determine the tier classification a laboratory falls under. If a lab
has averaged 3 or more proficiency tests and 1 or more certifications, then that lab is classified as a Tier 1. Those
averaging below those numbers are designated as Tier 2. An annual review of Tier classifications will take place
before the billing cycle begins. The laboratory will be notified by CDC and APHL in writing of the current tier
status prior to billing. Laboratories will be billed by APHL within the first weeks of the yearly cycle. If a new
laboratory joins PulseNet mid billing cycle, charges will be determined by CDC and APHL.

PulseNet Customized MasterScripts

As stated in the PulseNet SOP PNG05, Duties and Responsibilities of a PulseNet Laboratory, participating
laboratories are expected to be using the latest version of the PulseNet MasterScripts. PulseNet MasterScripts are
$1,000 per laboratory upon joining the network and an additional $1,000 for any future major upgrade. Laboratories
will be notified by CDC and APHL when major upgrades have been made. APHL will bill laboratories once the
MasterScripts have been distributed.

PulseNet Training

PulseNet Training is $250 per laboratorian per course. Training opportunities are announced on the PulseNet
SharePoint site as information becomes available. All training course applicants are considered on an as-needed
basis, charges will not be incurred if an applicant is not accepted into the course.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
APPENDIX PNG08-2

MEMORANDUM OF UNDERSTANDING
Between:

National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Centers for Disease Control and
Prevention (CDC)
and
Participating PulseNet USA Public Health and State Agricultural Laboratories

WHEREAS The Enteric Diseases Laboratory Branch (EDLB), Division of Foodborne, Waterborne and
Environmental Diseases, NCEZID, Centers for Disease Control and Prevention (CDC) relies on the participation of
state, county, and local public health and state agricultural laboratories in PulseNet USA, a national network
dedicated to the laboratory surveillance of enteric pathogens using standardized molecular subtyping methods; and

WHEREAS state, county, and local public health and state agricultural laboratories in the United States rely on the
leadership of EDLB for participation in a network dedicated to the laboratory surveillance of enteric pathogens;

NOW THEREFORE NCEZID and participating state, county, and local PulseNet laboratories agree that it is in
each of their interests to develop a joint cooperative program, namely the PulseNet USA network, for surveillance of
enteric pathogens.

1. INTRODUCTION
1.1 This Memorandum of Understanding (MOU) between selected state, county, and local public health and state
agricultural laboratories and NCEZID, hereinafter collectively referred to as the “Participating Laboratories,”
describes an arrangement to be willingly entered into by the Participating Laboratories. “Participants” refers
to the individuals at the participating laboratories who perform PulseNet associated functions.
1.2 This MOU is not legally binding and places no legal obligation on the Participating Laboratories.

2. OBJECTIVES AND SCOPE

2.1 The objective of this MOU is to establish an understanding among the Participating Laboratories concerning
their respective roles and responsibilities and to provide general guidelines under which cooperative activities
may be implemented.
2.2 The purpose of any proposed joint program is to support collaborative activities related to the PulseNet USA
enteric pathogens surveillance network.

3. ROLES AND RESPONSIBILITIES

3.1 Joint programs and projects will be consistent with the needs, merits, and goals of established programs within
PulseNet USA (CDC, NCEZID and/or the state, county, and local public health laboratories). In general, the
cooperative efforts described herein, and other joint cooperative programs and projects among the
Participating Laboratories, may be initiated at any time that a specific agreement can be reached involving
content, responsibility, liability, funding, intellectual property and other appropriate provisions, provided that
the process is consistent with the administrative regulations and procedures of the Participating Laboratories.
3.2 The Participating Laboratories agree to comply, to the best of their ability, with the attached Terms of
Reference (TOR), which details the collaborative activities of the Participating Laboratories and Participants.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
3.3 The following cooperative activity areas have been identified:
3.3.1 Exchange information that is not publicly available, to the extent allowed by U.S. law and other
Participating Laboratories’/Participants’ governing law;
3.3.2 Coordinate and collaborate on public health activities related to foodborne disease investigation;
3.3.3 Share protocols, standards, strains and human resources;
3.3.4 Participate in, coordinate, and implement quality assurance and quality control programs.
4. FINANCIAL ARRANGEMENTS
4.1 No funds are authorized or guaranteed under this MOU. Qualified public health laboratories have received
and may continue to receive support through the Epidemiology and Laboratory Capacity cooperative
agreement mechanism and/or the Emerging Infections Program. It is recognized that all Participating
Laboratories will, subject to the availability of appropriations, commit resources to keep PulseNet USA
operational.
5. SETTLEMENT OF DISPUTES
5.1 Any disputes regarding the interpretation or implementation of this MOU will be resolved only by consultation
between the Participating Laboratories and will not be referred to any third party for settlement.
6. AMENDMENT
6.1 This MOU may be amended by consent of all Participating Laboratories who have signed the MOU upon
receipt of written notice.
7. DURATION AND TERMINATION
7.1 This MOU will remain in effect until further notice. It shall be reviewed and renewed at least every three (3)
years by NCEZID and the Participating Laboratories.
7.2 This MOU may be terminated by any Participating Laboratories at any time provided that a 90 day written
notice is given to the other Participating Laboratories and appropriate steps are taken to ensure an orderly
termination of joint activities.
8. POINTS OF CONTACT

The National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and
Prevention
Administrative
Name: Dr. Peter Gerner-Smidt
Title: Chief PulseNet USA
Phone: 404 639 3322
Fax: 404 639 3333
e-mail: [email protected]

Technical (laboratory methods)

Name: Dr. Efrain Ribot
Title: Chief PulseNet Methods Development and Validation Laboratory
Phone: 404 639 3521

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
Fax: 404 639 3333
e-mail: [email protected]

Technical (database)
Name: Kelley Hise
Title: Chief Database Team
Phone: 404 639 0704
Fax: 404 639 3333
e-mail: [email protected]

Technical (software and database security)

Name: Brenda L. Brown
Title: PulseNet Technical and Security Steward
Phone: 404 639 3942
Fax: 404 639 3333
e-mail: [email protected]

Participating state, county or local public health or state agricultural laboratory

Administrative
Name:
Title:
Phone:
Fax:
e-mail:

Technical
Name:
Title:
Phone:
Fax:
e-mail:

9. EFFECTIVE DATE AND SIGNATURE

9.1 The effectiveness of this MOU does not depend upon the signature of every PulseNet USA Participating
Laboratory. This MOU becomes effective when two of the Participating Laboratories have signed the MOU,
upon the date of the later signature. The MOU will be effective only among the Participating Laboratories that
have signed the MOU.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
The National Center for Emerging and Zoonotic Infectious Diseases,
Centers for Disease Control and Prevention

__________________________________
Dr. Beth Bell MD MPH, Director, NCEZID

__________________________
Date

Participating state, county or local public health laboratory

___________________________________
Name of laboratory

___________________________________
( )

__________________________________
Date

Reviewed by
___________________________________
Name of State Epidemiologist

___________________________________
( )

__________________________________
Date

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
APPENDIX PNG08-3
Terms of Reference for PulseNet USA

1. SCOPE: The National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Centers for

Disease Control and Prevention (CDC) and the participating PulseNet USA state, county, and local

laboratories agree that it is in their mutual interest to develop a joint cooperative program to perform

standardized DNA fingerprinting of foodborne disease-causing bacteria so that the participating

organizations will be able to rapidly compare the DNA fingerprints of infectious agents from any

laboratory participating in the PulseNet network. This will facilitate rapid and early identification of

disease clusters and disease outbreaks at the national level and, when coordinated with PulseNet

International networks, will serve as an effective global early alert system for foodborne disease outbreaks.

The PulseNet network may be extended to cover surveillance for other pathogens.

2. PURPOSE: This Terms of Reference document (TOR) will define the rights and responsibilities of each

participant in the PulseNet USA network.

3. DEFINITIONS: The following terms/abbreviations will be used throughout the document:

3.1 DNA fingerprint – A subtype result obtained by a standardized DNA molecular method that has

been adopted by the PulseNet network.

3.2 TOR: Terms of Reference

3.3 Participating Laboratory: A federal, state, county, or local public health, or state agricultural

laboratory that engages in PulseNet activities.

3.4 Participant: An individual at a Participating Laboratory who engages in PulseNet activities.

3.5 PulseNet USA Steering Committee – A committee made up of selected members of the PulseNet

USA network, from both CDC and state, county, or local laboratories and APHL.

4. BACKGROUND: Foodborne infections are a national and global problem. Trade of raw and processed

food across borders within and between different regions of the world and international travel make it

possible that the source of a foodborne infection may be located in a different country, state, or region than

where the illnesses are observed. International trade and travel has grown rapidly during the past decades;

consequently, foodborne infections increasingly show up in parts of the world differing from their origin.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
A critical component in the investigation of foodborne outbreaks is the DNA fingerprinting of the causative

organisms and comparison of the DNA fingerprints of strains isolated from ill persons who are considered to be part

of a common source outbreak and the possible sources throughout the food chain. At present, Pulsed-Field Gel

Electrophoresis (PFGE) is the gold standard routine method for comparison of most bacterial foodborne pathogens.

CDC began setting up the PulseNet USA network in 1996 to facilitate rapid, standardized DNA fingerprinting of

foodborne pathogens by state and local public health laboratories in the United States and their submission to a

central electronic database at CDC for purposes of making the database of fingerprints available to PulseNet

participants. Currently, PulseNet standardized protocols are available for Shiga toxin (Verocytotoxin) producing E.

coli (STEC) O157:H7, non-O157 STEC, Salmonella, Listeria monocytogenes, Shigella species, Shigella flexneri,

Vibrio cholerae, Vibrio parahaemolyticus, Clostridium perfringens, Clostridium botulinum, Yersinia pestis and

Campylobacter species. Next generation subtyping tools, e.g., Multi Locus VNTR Analysis (MLVA) has been

implemented or is under development and validation for STEC O157, Salmonella serovars Enteritidis and

Typhimurium and Listeria monocytogenes. More recently, PulseNet has begun exploring the use of whole genome

sequencing (WGS) as a next-generation subtyping tool. The plan to use this technology has initiated a review of

PulseNet data disclosure policy: optimal sharing of WGS data and a subset of associated metadata involves public

disclosure while protecting patient confidentiality and avoiding inappropriate use of preliminary or potentially

sensitive public health data. . The primary drivers and benefits to the public release of a subset of data include:

1) To conform with the President’s Open Government Directive and the CDC/ATSDR Policy on Releasing and

Sharing Data

2) To provide basic information on the frequency and genotypes of foodborne pathogens infecting people to the

food industry, so they can potentially use this information in combination with their information to assess

the possible risk posed by their products.

3) To provide researchers in microbial phylogenetics, molecular epidemiology and food microbiology access to

data useful in their research endeavors.

In addition, the amount of sequence data potentially produced in PulseNet will require collaboration with partners

with the capacity to store and manage extremely large datasets. This is accomplished in collaboration with the

National Center for Biotechnology Information (NCBI). Data exchange may occur in the Sequence Read Archieve

VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
(SRA) in the Genbank database. The metadata to be shared with the WGS data is presented in more detail in Section

6.B.4.

The PulseNet network has enabled CDC and its public health partners to detect and investigate outbreaks of

foodborne infections in humans in the United States as well as international outbreaks in cooperation with public

health colleagues in PulseNet International.

The objective of PulseNet USA is to use DNA fingerprinting to characterize foodborne pathogens in order to combat

national and global outbreaks and to perform surveillance of the different foodborne pathogens throughout the food

chain and of the infections they cause in humans. An ultimate goal is to help decision makers in establishing policies

for safer food on the national and global level.

5. GOVERNANCE:

5.1 Activities of the PulseNet USA network are coordinated by the PulseNet USA Steering Committee. The

PulseNet USA Steering Committee is chaired by the chief of the Enteric Diseases Laboratory Branch

(EDLB) or his substitute at CDC and is comprised of participants from state and local PulseNet

Laboratories, APHL and CDC.

5.2 Membership on the PulseNet USA Steering Committee is by invitation; terms on the Committee are

open-ended. The Committee meets via conference call as necessary and generally once per quarter.

The structure and membership of the committee may be changed upon agreement of the current

members.

5.3 Decisions regarding topics brought before the committee are reached by consensus.

6. ROLES AND RESPONSIBILITIES OF PARTICIPANTS:

A. Coordination and collaboration relative to public health activities

It is mutually agreed that:

6.A.1 Each participating laboratory will utilize the expertise, resources, and relationships of the network in

order to increase capability and readiness to respond to outbreaks. In addition, each participating laboratory

will designate central contact points where communications dealing with matters covered by this agreement

should be referred.

6.A.2 One or more participants from each participating laboratory will attend periodic joint meetings to

VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
promote better communication and understanding of regulations, policies, and responsibilities and to address

questions and issues that may arise through routine or critical operation of the network.

6.A.3 One or more participants at each participating laboratory will maintain knowledge of improvements to

the communication methods, data utilization tools, and methodological developments within the network.

6.A.4 Each participating laboratory will notify the other participating laboratories as soon as possible when

issues of mutual concern become evident. 6.A.5 Each participating laboratory will collaborate with the

other participating laboratories in all investigations of mutual concern. Such collaboration may include

providing alerts to the other participating laboratories regarding disease outbreaks encountered as part of its

activities, providing technical advice in areas of recognized expertise, providing results of analysis, and

exchanging information, e.g. identification at the state or local level of indistinguishable DNA fingerprints,

developments of a subtyping method that potentially may be implemented in the network or some legal

changes at the local level which may have consequences for the network as a whole.

6.A.6 Each participating laboratory will keep all information received from other participants confidential

unless the information is already publicly available or written consent for distribution has been received from

the originating laboratory.

6.A.7 This agreement does not preclude the participating laboratories and/or participants from entering into

other agreements which may set forth procedures for special programs which can be handled more efficiently

and expertly by other agreements.

B. Principles and Procedures for the Exchange of Information that is not Publicly Available

It is mutually agreed that:

6.B.1 Although there is no legal requirement for exchange of information, the PulseNet participating

laboratories/participants agree in principle that there should be a presumption in favor of full and free sharing

of information between them. The participants recognize and acknowledge, however, that it is essential that

any confidential information that is shared between them must be protected from unauthorized public

disclosure. Safeguards are important to protect the interests of, among others, owners and submitters of trade

secrets and confidential commercial information, patient identities and other personal privacy information,

privileged and/or pre-decisional agency records, and information protected for national security reasons.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 14 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
Such safeguards also help guarantee the participant's compliance with applicable laws and regulations. A

participant should only decide not to share information if credible information exists that the requesting

participant may not be able to comply with applicable laws or regulations governing the protection of non-

public information or with the principles or procedures set forth in this TOR.

6.B.2 All participants must implement appropriate data and information security systems. To facilitate the

sharing of information, the participants must implement procedures to ensure, at a minimum, that such

sharing of information is indeed appropriate and that the recipient guards the confidentiality of all

information received. Document control procedures should also be implemented for the storage of any hard

copy print outs of PulseNet data.

6.B.3 All PulseNet USA participating laboratories/participants shall limit the dissemination of shared

information to those participants, internal agency offices, and/or individuals that reasonably require the

information and will be responsible for ensuring that there are no other recipients of the information.

6.B.4 Sharing of whole genome sequence (WGS) data and a subset of associated metadata optimally

involves public disclosure while protecting patient confidentiality and avoiding inappropriate use of

preliminary or potentially sensitive public health data. In order to protect patient confidentiality the patient

specific data to be released publically will be limited to clinical specimen type yielding the pathogen (i.e.,

blood, stool) the year of illness onset and region of country for patient residence (i.e., HHS region, see

Appendix 1). To avoid inappropriate use of preliminary or potentially sensitive information, release of

patient data will occur six months after the release of isolate data. Specifically, an isolate identifier, a

taxonomic description of the isolate (genus and species), the source (i.e., clinical, food, or environmental),

the country of origin along with the sequence information will be released to Genbank as soon as WGS data

is available. After six months and if approved by the participant, information on isolation site of the

organism, the serotype of the organism (if applicable),the year of isolation, the geographic region of patient

residence (HHS region), and the age group of the patient will be released.

Each PulseNet participating laboratory must indicate by checking off the appropriate box and signing the

form (Appendix 2) attached to this TOR by an appropriate official if the delayed metadata may be released.

6.B.5 All PulseNet USA participants shall keep any shared information confidential, to the extent permitted

VERSION: REPLACED BY: AUTHORIZED BY:

Page 15 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
by law, until otherwise agreed upon.

C. Protocols, Standards, Reference Strains and Quality Assurance

6.C.1 Each participant agrees to follow the standardized PulseNet protocol for subtyping of foodborne

isolates, as distributed and/or published by CDC.

6.C.2 Each participant agrees to utilize the customized PulseNet scripts for analysis of PFGE patterns and

other PulseNet subtyping data, assisted by the recommended version of the analysis software and according

to the analysis guidelines distributed by CDC.

6.C.3 XbaI digested DNA of the strain Salmonella Braenderup H9812, provided to PulseNet networks

participants by CDC and available through the American Type Culture Collection (ATCC number BAA-

664), is the universal molecular size standard against which all PFGE profiles generated in the networks are

normalized.

6.C.4 Each participating laboratory should establish a culture collection containing strains representing

each unique pattern in their databases. The participants agree to share these strains with each other for

network purposes at no cost or at the cost of shipping and handling.

6.C.5 Each participating laboratory agrees to participate in certification and proficiency testing programs

designed to ensure comparability of profiles between the participants.

7. PULSENET NAME AND LOGO:

7.1 The PulseNet name and logo are trademarks that are owned by CDC. Signatories to this TOR may use

the PulseNet name as needed for use in PulseNet USA activities. However, because the PulseNet logo

contains the CDC logo signatories must obtain specific CDC approval before using the PulseNet logo.

7.2 When a participating laboratory exercises its option to terminate the PulseNet USA MOU, that

participating laboratory immediately loses the right to use the PulseNet name and logo.

8. SYSTEMS AND DATA SECURITY MEASURES

8.1 All participants will install and configure system equipment capable of running the latest recommended

version of the PulseNet Customized software. Data security will be maintained following the direction of

the PulseNet Technical and Security Steward, including such mechanisms as storage in a locked room or

locked file cabinet, shredding of discarded documents, and memory erasure upon replacement of hard

VERSION: REPLACED BY: AUTHORIZED BY:

Page 16 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
drives. Participants will not share logon identities, passwords, or SecurID devices that have been assigned

to them by CDC.

8.2 All participants who have access to the PulseNet listserv (SharePoint or the most current

communication mechanism) will sign a non-disclosure statement assuring that all information will be

treated as confidential and only shared with appropriate public health personnel or others as may be

required by law.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 17 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13

Appendix 1. HHS regions (from http://www.hhs.gov/about/regionmap.html)

• Region 1 - Boston • Region 6 - Dallas

Connecticut, Maine, Massachusetts, New Arkansas, Louisiana, New Mexico, Oklahoma,
Hampshire, Rhode Island, and Vermont and Texas
• Region 2 - New York • Region 7 - Kansas City
New Jersey, New York, Puerto Rico, and the Iowa, Kansas, Missouri, and Nebraska
Virgin Islands • Region 8 - Denver
• Region 3 - Philadelphia Colorado, Montana, North Dakota, South Dakota,
Delaware, District of Columbia, Maryland, Utah, and Wyoming
Pennsylvania, Virginia, and West Virginia • Region 9 - San Francisco
• Region 4 - Atlanta Arizona, California, Hawaii, Nevada, American
Alabama, Florida, Georgia, Kentucky, Samoa, Commonwealth of the Northern Mariana
Mississippi, North Carolina, South Carolina, Islands, Federated States of Micronesia, Guam,
and Tennessee Marshall Islands, and Republic of Palau
• Region 5 - Chicago • Region 10 - Seattle
Illinois, Indiana, Michigan, Minnesota, Ohio, Alaska, Idaho, Oregon, and Washington
and Wisconsin

VERSION: REPLACED BY: AUTHORIZED BY:

Page 18 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13

Appendix 2

Metadata to be released immediately to NCBI’s Genbank with a whole genome sequence (WGS) and with a 6

months delay following the upload of the sequence.

Metadata released immediately with the WGS:

• Unique sample ID (WGS_ID)
– Created specifically for this purpose in order NOT to include information about isolation date or
state of origin
• Organism genus and species (e.g., Listeria monocytogenes)
• Organism source (e.g., clinical, food, environment)
• Country of origin (USA)
• ID for laboratory submitting DNA sequence (e.g., CDC)
Metadata for release delayed 6 months* after upload of WGS:
• Site of isolation (e.g., blood, cerebral spinal fluid, stool)
• Organism serotype (if applicable and available)
• Collection year
• Geographic location (HHS Region of patient residence)
• Age category (in years: 0-4, 5-9, 10-19, 20- 29, 30- 39, 40- 49, 50- 59, 60- 69, 70- 79, 80+)
* Six- seven months will be the usual period of delay; the update of metadata in GenBank should occur on a

monthly basis.

___ Metadata may be submitted as described above for immediate and delayed release
___ Only metadata for immediate release as described above may be released
(Check one)

Signature Date

__________________________________________________________________

Name, Affiliation, PulseNet laboratory

VERSION: REPLACED BY: AUTHORIZED BY:

Page 19 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
APPENDIX PNG08-4

Welcome E-mail

Greetings [Lab Director],

Welcome to PulseNet! We are excited about your laboratory’s interest in PulseNet and pleased to include
the [XXXX] lab in our constantly expanding network of participants. Please feel free to contact us with
any questions.

We are looking forward to working with your laboratory!

Sincerely,

Kelley Hise, MPH Kristy Kubota, MPH

Chief, PulseNet Database Unit Senior Specialist, PulseNet Program
Centers for Disease Control and Prevention Association of Public Health Laboratories
1600 Clifton Rd. NE 8515 Georgia Avenue
MS C-03 Suite 700
Atlanta, GA 30329 Silver Spring, MD 20910
[email protected] [email protected]
Phone: (404) 639-4558 Phone: (240) 485-2720
Fax: (404) 639-3333 Fax: (240) 485-2700

VERSION: REPLACED BY: AUTHORIZED BY:

Page 20 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
APPENDIX PNG08-5

Welcome Letter for New PulseNet Laboratories

[Date]

[Lab Director]
[Lab Address/Information]

Welcome to PulseNet! We are excited about your laboratory’s interest in PulseNet and pleased to include
the [XXXX] lab in our constantly expanding network of participants. As you read through the documents
provided in this packet, along with those posted in the Library of PulseNet Documents on PulseNet’s
SharePoint site, please do not hesitate to contact CDC, APHL or your Area Laboratory with any
questions.

Your PulseNet LabID is [XXXX]. You will need this LabID to install the PulseNet MasterScripts and also
use it to name all TIFF and bundle files created in your lab.

Your PulseNet Area Laboratory is [XXXX, contact info]

Information provided in this packet for your reference:

 Standard Operating Procedures (SOPs) included in this packet
 PNG05 Responsibilities of a PulseNet Laboratory
 PND03 PulseNet SharePoint, Access and Use
 PNQ02 Certification
 “Tips for Getting Started with PulseNet” is a document that was created to help answer some
commonly asked questions
 PulseNet organisms and their differences

We look forward to working with you.

Kelley Hise, MPH Kristy Kubota, MPH

Chief, PulseNet Database Unit Senior Specialist, PulseNet Program
Centers for Disease Control and Prevention Association of Public Health Laboratories
1600 Clifton Rd. NE 8515 Georgia Avenue
MS C-03 Suite 700
Atlanta, GA 30333 Silver Spring, MD 20910
[email protected] [email protected]
Phone: (404) 639-4558 Phone: (240) 485-2720
Fax: (404) 639-3333 Fax: (240) 485-2700
Website: www.cdc.gov/pulsenet Website: www.aphl.org

VERSION: REPLACED BY: AUTHORIZED BY:

Page 21 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
APPENDIX PNG08-6

Tips for Getting Started with PulseNet

 General
 Contact Information
o PulseNet Database Team at CDC (404) 639-4558 or [email protected]
 Please always use [email protected] when contacting CDC via email. Your emails will
automatically be directed to the appropriate person. Using this email address will ensure a
more prompt response in case you do not know who to contact for a specific question,
someone is out of the office or no longer works at CDC.
o Kristy Kubota, Senior Specialist, PulseNet Program at APHL (240) 485-2720 or
[email protected]
 There are currently eight PulseNet Area Laboratories. These are labs, designated by CDC and
APHL that have agreed to assume responsibility for additional PulseNet duties for laboratories
within their support region. The current Area Labs include MA, MN, WA, TX, VA, UT, MI and
CDC. Refer to SOP PNG06 for more specific information on Area Lab responsibilities and how
they may assist you.
 Websites
o PulseNet www.cdc.gov/pulsenet
o APHL www.aphl.org
 Visit APHL’s website and click on “Conferences” at the top of the page to find
information on the InFORM Conference
 The PulseNet SharePoint site houses important PulseNet information including cluster and
outbreak information, procedures, training documents, tips and reminders, and many more useful
references. To request access to SharePoint, send an email to [email protected] with “SharePoint”
in the subject line. Include your name, email, and position within the laboratory. Refer to SOP
PND03 for more information, including how your epidemiologists may receive access.
 Watch SharePoint postings for lab and software training opportunities at CDC. If no training
courses are being advertised at the time, you may contact your Area Laboratory and/or CDC to
discuss other possible opportunities.
 Laboratory
 PFGE protocols are available on SharePoint within the “Library of PulseNet Documents/QA/QC
Manual” section and on the PulseNet website www.cdc.gov/pulsenet
 Troubleshooting advice is available within the troubleshooting discussions on SharePoint.
Laboratories are also encouraged to contact their Area Laboratory and/or CDC for
troubleshooting assistance.
 Database
 A separate database must be created for each PulseNet organism run in your laboratory. Each
organism is PFGE’d under different running conditions, therefore the reference standard
(Salmonella Braenderup H9812) patterns look different. To take the different reference standard
patterns into account, the PulseNet MasterScripts vary by organism and must only be run on the
appropriate databases. If you ever need assistance with database setup, please contact CDC.
 Laboratories are assigned a LabID upon joining PulseNet. Your LabID must be entered to run
PulseNet MasterScripts in BioNumerics databases and used to name TIFF and bundle files
submitted to PulseNet.
 Name all files according to the standardized PulseNet naming system. The first two to four
characters should be the LabID (i.e. CDC), the second two digits should be the year (i.e. 14), and
VERSION: REPLACED BY: AUTHORIZED BY:

Page 22 of 23
 STANDARD OPERATING PROCEDURE FOR BECOMING A PULSENET CODE: PNG08
PARTICIPATING LABORATORY AND HANDLING REQUESTS TO BECOME A Effective Date:
PARTICIPATING LABORATORY 01 02 13
the last three digits should be the unique number of the TIFF or bundle file submitted that year
from your laboratory (i.e. 0001).
 QA/QC
 The PulseNet Standard Operating Procedures (SOPs) are provided on SharePoint within the
“Library of PulseNet Documents/QA/QC Manual” and may be downloaded and/or printed for use
in your laboratory. The SOPs are broken into four categories: General, Laboratory, Database, and
QA/QC, to help locate procedures easily. For example, all of the PFGE laboratory protocols can
be found within the Laboratory section.
 Certification
o Once a person is routinely producing good quality PFGE images, they may complete and
submit for PFGE gel certification.
o Once a person is comfortable with PFGE analysis and the creation of PulseNet bundle files in
BioNumerics, they may complete and submit analysis certification.
o Certification sets are available for Salmonella, E. coli O157, Non O157 STEC, Shigella,
Shigella flexneri, Listeria monocytogenes, Campylobacter jejuni, Vibrio cholerae, Vibrio
parahaemolyticus and C. botulinum. These sets may be requested by emailing
[email protected] with “Certification” in the subject line. Participants may become gel-
certified, analysis-certified, or both for each organism. Participants must be analysis-certified
in an organism to receive access to that specific national database. Laboratories must have at
least one person gel-certified in a specific organism before anyone in the lab may be analysis-
certified for that organism.
 Laboratories must participate and successfully complete annual proficiency testing (PT) to retain
certification status for each organism.
o Currently PT for Salmonella, E. coli, and Shigella is held in the Fall and Campylobacter,
Listeria and Vibrio are held in the Spring.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 23 of 23
READING CONTROL SHEET FOR:
GENERAL STANDARD OPERATING PROCEDURES (PNG)

NAME DATE COMMENTS SIGNATURE

By signing above, you are indicating that you have read and understood all SOPs
included in the PNG section of this manual.
READING CONTROL SHEET FOR:
STANDARD OPERATING PROCEDURES FOR THE PULSENET LABORATORY
(PNL)

NAME DATE COMMENTS SIGNATURE

By signing above, you are indicating that you have read and understood all SOPs
included in the PNL section of this manual.
 STANDARD OPERATING PROCEDURE FOR LABORATORY EQUIPMENT AND CODE: PNL01
Effective Date:
SUPPLIES 5 09 2005

1. PURPOSE: To describe the laboratory equipment and supplies needed for molecular subtyping of
foodborne bacterial pathogens by Pulsed-field Gel Electrophoresis (PFGE).

2. SCOPE: This procedure applies to all PulseNet participating laboratories performing PFGE.

3. DEFINITIONS/TERMS:

3.1. PFGE: Pulsed-field Gel Electrophoresis

3.2. TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
3.3. BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium

4. RESPONSIBILITIES/PROCEDURE:

Electrophoresis and Documentation Equipment:

Bio-Rad CHEF Mapper XA System, CHEF-DR III Variable Angle System, or GenePath Strain
Typing System with PC customized software program. Include pump and chiller module.

Bio-Rad Gel Doc 2000 or ChemiDoc Documentation System

or
An equivalent documentation system that is equipped with a CCD camera that can provide IBM-
compatible uncompressed TIFF images and resolution of ≥768 x 640 pixels, and which will allow
comparison of images in the PulseNet database with BioNumerics software (Applied Maths,
Inc.).

Other Equipment:

37ΕC Incubator (or appropriate temperature) - incubate cultures

Dade Microscan Turbidity Meter1, Spectrophotometer, or bioMérieux Vitek Colorimeter

- adjust concentration of cell suspensions

Microwave - melt agarose

Water Bath with Shaker or Shaking Incubator

- lyse cells in agarose plugs (54°C)
- wash plugs with water and TE (50°C)

56°C Water Bath2 - equilibrate and hold melted agarose

25°C, 30°C, 37°C Water Bath(s) - restriction digestion reactions
50°C Water Bath - heat water and TE that is used to wash plugs; restriction digestion reactions

Microcentrifuge - briefly centrifuge small vials of reagents such as restriction digestion buffer,
Proteinase K, and other enzymes

1
Dade Behring, Inc., 1717 Deerfield Rd. Deerfield, IL 60015
2
At a minimum, two water baths are needed – one equilibrated to 56°C and one to 37°C. Temperature can be
increased or decreased as needed. ApaI restriction of Listeria PFGE plugs slices requires a 30°C water bath.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 4
STANDARD OPERATING PROCEDURE FOR LABORATORY EQUIPMENT AND CODE: PNL01
Effective Date:
SUPPLIES 5 09 2005

Supplies and Selected Reagents:

SeaKem Gold Agarose (Cambrex Bio Sciences Rockland, Inc. 50152, 50150
- casting plugs in reusable or disposable plug molds
- PFGE gel

Sterile Ultrapure H2O (Reagent Grade, Type 1)

Proteinase K Powder
Amresco - 0706
Invitrogen [Life Technologies] - 25530-031
Roche Molecular Biochemicals - 1 000 144, 1 092 766
or
Proteinase K Liquid
Amresco - E195
Invitrogen [Life Technologies] - 25530-049
Roche Molecular Biochemicals - 1 964 372, 1 964 399

5X TBE - dilute 1:10 to make 0.5X TBE

Amresco - J885
Sigma/Aldrich - T6400
or
10X TBE - dilute 1:20 to make 0.5X TBE
Amresco - 0658
Bio-Rad - 161-0733, 161-770
Invitrogen [Life Technologies] - 15581-044
Roche Molecular Biochemicals - 100 759, 1666 703
Sigma - T4415

Agar plates and slants

Trypticase Soy Agar with 5% defibrinated sheep blood (TSA-SB), Heart Infusion Agar (HIA),
or TSA - E. coli, Salmonella, and Shigella
TSA-SB, Brain Heart Infusion Agar (BHIA) - Listeria monocytogenes
HIA with Rabbit Blood - Campylobacter jejuni, C. coli

Restriction Enzymes and Appropriate Buffers (Roche Molecular Biochemicals, New England
Biolabs, Promega, or other supplier) for each organism tested.

E. coli O157:H7, Salmonella, Shigella sonnei

XbaI, AvrII (isoschizomer BlnI), SpeI
(NotI for S. sonnei; do not use for E. coli O157:H7)
L. monocytogenes
AscI, ApaI
Campylobacter jejuni, C. coli
SmaI, KpnI

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 4
 STANDARD OPERATING PROCEDURE FOR LABORATORY EQUIPMENT AND CODE: PNL01
Effective Date:
SUPPLIES 5 09 2005

PulseNet Standard Strain

Salmonella ser. Braenderup H9812 and PFGE plugs of H98123

DNA Size Standard, Lambda Ladder

Bio-Rad - 170-3635
Cambrex Bio Sciences Rockland, Inc. - 50461
Roche Molecular Biochemicals - 1378 961, or other supplier
Ethidium bromide (Amresco - X328; Bio-Rad - 161-0433; Sigma - E-1510)

Miscellaneous Supplies:

Sterile clear polystyrene 12-mm x 75-mm (Falcon 2054) or 17-mm x 100-mm tubes
(Falcon 2057) tubes with cap (or equivalent) - for cell suspensions

Sterile polyester-fiber or cotton swabs - remove growth from agar plates

Sterile transfer pipets or sterile Pasteur pipets and rubber bulbs - use when adjusting cell
suspensions or to remove reagents from plug slices

Sterile 1.5 ml microcentrifuge tubes - mix cell suspensions with agarose; restriction digestions

Sterile 50 ml polypropylene screw-cap tubes (Falcon 2098, Corning 25330-50) or

50 ml Oak Ridge tubes (Nalgene 3118-50) - for plugs made in reusable or disposable molds

Green Screened Caps, Bio-Rad 17037114 - use when washing PFGE plugs

PFGE plug molds - 10-well reusable (2-cm x 1-cm x 1.5-mm; Bio-Rad 170-3622)
or
- 50-well disposable (1.5-mm x 10-mm x 5-mm, Bio-Rad 170-3713)

Single-edge razor blades, scalpels, glass cover slips, or equivalent - use to cut plug slices

Sterile disposable petri dishes or large glass slides - can use when cutting plugs

Flat spatulas with one wide and one tapered end

Standard Casting Stand (14 x 13 cm frame and platform) - Bio-Rad 170-3689

Wide/Long Combination Casting Stand (21 x 14 cm frame and platform) - Bio-Rad 170 -3704

Combination Comb Holder - Bio-Rad 170-3699

10-well Comb, 14 cm long, 1.5 mm wide - Bio-Rad 170-4326

3
S. ser. Braenderup H9812 restricted with XbaI will be used as the standard strain for all organisms tested by
PulseNet after March 15, 2003.
4
These caps will not fit the 50 ml Oak Ridge tubes.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 4
 STANDARD OPERATING PROCEDURE FOR LABORATORY EQUIPMENT AND CODE: PNL01
Effective Date:
SUPPLIES 5 09 2005

15-well Comb, 21 cm long, 1.5 mm wide - Bio-Rad 170-3627

Gel-leveling table

Plastic containers for staining gels with ethidium bromide

70% isopropanol, bleach (5% - 10%), or other appropriate disinfectant

Sterile screw cap flasks or bottles of various sizes (50 ml - 2000 ml)

Sterile graduated cylinders of various sizes (100 ml - 2000 ml)

Single-channel micropipetters - fixed and/or variable volume of various sizes

Sterile pipets (2 ml - 50 ml) and pipet tips (10 Φl - 1000 Φl)

Protective gloves (powder-free latex, vinyl, or nitrile)

Heat-resistant gloves

Ice Bucket

Use of trade names and commercial sources is for identification only and does not imply
endorsement by the CDC or the U.S. Department of Health and Human Services.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 4
STANDARD OPERATING PROCEDURE FOR PREPARATION OF REAGENTS USED CODE: PNL02
Effective Date:
IN THE PULSENET STANDARDIZED PFGE PROTOCOLS 08 14 2013

1. PURPOSE: To describe the formulas of stock reagents used for standardized PulseNet Pulsed-field Gel
Electrophoresis (PFGE) protocols of foodborne bacterial pathogens.

2. SCOPE: To describe the procedures for preparation of reagents used in the PulseNet standardized PFGE
protocols to be used by the PulseNet participants in order to assure inter-laboratory comparability of results
generated.

3. DEFINITIONS/TERMS:
3.1 PFGE: Pulsed-field Gel Electrophoresis
3.2 DNA: Deoxyribonucleic acid

4. RESPONSIBILITIES/PROCEDURE:

Sterile Ultrapure or Reagent Grade Type 1 (NCCLS) Water* 1

Autoclave or filter-sterilize in 100 ml, 500 ml, and/or 1 liter volumes in screw-cap bottles or flasks.

1 M Tris≅HCl, pH 8.0*

121.1 g Tris base

Dissolve in 650-700 ml Ultrapure H2O
Add 80 ml 6 N HCl*
Let solution come to room temperature
Make final adjustments to pH
Dilute to 1000 ml with Ultrapure H2O
Sterilize by autoclaving
or
157.6 g Tris-HCl
Dissolve in 800 ml Ultrapure H2O
Let solution come to room temperature
Make final adjustments to pH
Dilute to 1000 ml with Ultrapure H2O
Sterilize by autoclaving

10 N NaOH*

400 g NaOH
Carefully dissolve in 800 ml sterile Ultrapure H2O
Cool solution to room temperature
Dilute to 1000 ml with sterile Ultrapure H2O

0.5 M EDTA, pH 8.0*

186.1 g Na2EDTA≅2H2O
Add 800 ml Ultrapure H2O

1
Reagents or chemicals marked with * are available from commercial companies such as Amresco, Fisher,
Invitrogen (Life Technologies), Roche Molecular Biochemicals, Sigma, Mediatech, Inc. (CellGro), and others.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 5
STANDARD OPERATING PROCEDURE FOR PREPARATION OF REAGENTS USED CODE: PNL02
Effective Date:
IN THE PULSENET STANDARDIZED PFGE PROTOCOLS 08 14 2013

Mix and adjust pH to 8.0 with 50 ml 10 N NaOH*

Note: Add 10 N NaOH slowly to solution; check with pH meter.

Dilute to 1000 ml with Ultrapure H2O

Dispense into aliquots. Sterilize by autoclaving

Phosphate Buffered Saline (PBS), 0.01M, pH 7.2 or pH 7.4* - use to make Campylobacter cell suspensions.
The following formula for PBS is from Molecular Cloning - A Laboratory Manual by J. Sambrook and
D. Russell, 3rd edition.
137 mM NaCl (8 g)
2.7 mM KCl (0.2 g)
10 mM Na2HPO4 (1.44 g)
2 mM KH2PO4 (0.24 g)

Dissolve in 800 ml Ultrapure H2O

Mix and adjust pH to 7.2 or 7.4 with HCl
Adjust final volume to 1000 ml with H2O
Sterilize by autoclaving for 20 minutes at 15 lb/sq. in. on liquid cycle.
Store at room temperature

20mg/ml Lysozyme Stock Solution - use in Listeria monocytogenes standardized PFGE Protocol.

100 mg Lysozyme (Available from Sigma, L7651 or L6875)

5 ml TE Buffer
Mix and dispense in 200-250 μl volumes in 1.5 ml microcentrifuge tubes; store at -20°C

10mg/ml Lysozyme Stock Solution - use in Listeria monocytogenes standardized PFGE Protocol.

50 mg Lysozyme (Available from Sigma, L1667)

5 ml TE Buffer
Mix and dispense in 200-250 μl volumes in smaller microcentrifuge tubes; store at -20°C

20% Sodium Dodecyl Sulfate (SDS)* - use in Listeria standardized PFGE protocol.

20 g SDS
80 ml sterile Ultrapure H2O
Carefully add SDS to H2O in sterile container; dissolve by mixing gently and warming to
35°- 45°C.

10% Sodium Dodecyl Sulfate (SDS)* - use in Listeria standardized PFGE protocol.

10 g SDS
80 ml sterile Ultrapure H2O
Carefully add SDS to H2O in sterile container; dissolve by mixing gently and warming to
35°- 45°C.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 5
 STANDARD OPERATING PROCEDURE FOR LABORATORY FORMULAS AND CODE: PNL02
Effective Date:
REAGENTS 08 14 2013

20 mg/ml Proteinase K Stock Solution*

100 mg Proteinase K powder
5 ml sterile Ultrapure H2O
Mix and dispense in 500-600 μl volumes in 1.5 ml microcentrifuge tubes; store at -20°C.

10% N-Lauroylsarcosine, Sodium salt (Sarcosyl) 2

10 g Sarcosyl (Available from Sigma, L-9150)
90 ml sterile Ultrapure H2O
Carefully add Sarcosyl to H2O in sterile container; dissolve by mixing gently and warming to 50° - 60°C

Safety note: Wear a mask when weighing Lysozyme, SDS, Proteinase K, and Sarcosyl; avoid creating aerosols;
and wipe down balance and surrounding area after weighing.

10X Tris-Borate EDTA Buffer (TBE), pH .8.3*

0.9 M Tris base (108 g)
0.9 M Boric Acid (55 g)
0.02 M EDTA, pH 8.0 (40 ml 0.5 M)
Dilute to 1000 ml with sterile Ultrapure H2O
Sterilize by autoclaving

Note: Discard if buffer develops precipitate!

Note: The previous formula for TBE is from Molecular Cloning - A Laboratory Manual by J. Sambrook and D.
Russell, 3rd Edition. It is similar to the formulas used by several commercial suppliers (Amresco10X - 0658, 5X
- J885; Fisher 10X - BP1333-1, BP13334; Roche Molecular Biochemicals 10X - 100 661, 100 759, 1 666 703;
Sigma-Aldrich 10X - T4415); it differs from 10X TBE from Invitrogen (Life Technologies) as documented in
the formula below. These differences in formulation may affect the mobility of the DNA during electrophoresis.

Invitrogen 10X TBE Buffer, 15581-044 or 15581-028*

1.0 M Tris base
0.9 M Boric Acid
0.01 M EDTA
pH of 10X solution = 8.4 ∀ 0.1

Ethidium Bromide*
10 mg/ml stock solution
Dilute 1:10,000 with Ultrapure H2O (10 μl in 100 ml H2O).

Diluted Ethidium Bromide solution can be used for staining –up to 20 gels before discarding according
to safety guidelines of your institution. See Section 10 of the PulseNet PFGE manual for further
information.

2
This chemical can be added directly to the other ingredients in the cell lysis buffer. See page 5.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 5
 STANDARD OPERATING PROCEDURE FOR LABORATORY FORMULAS AND CODE: PNL02
Effective Date:
REAGENTS 08 14 2013

WORKING REAGENTS

Use sterile glassware, plasticware, and Ultrapure (Reagent Grade Type 1) Water to make working reagents.

Tris:EDTA Buffer (TE), pH 8.0*

10 mM Tris-HCL:1 mM EDTA , pH 8.0 3

10 ml 1 M Tris-HCL, pH 8.0
2 ml 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure H2O

E. coli O157:H7, Salmonella and Shigella sonnei, and Campylobacter jejuni standardized PulseNet PFGE
protocols - use this TE Buffer to:
a. Dissolve 1% SeaKem Gold
b. Dissolve 1% SeaKem Gold:0.5% SDS
c. Wash PFGE plugs after cell lysis.

L. monocytogenes standardized PulseNet PFGE protocol - use this TE Buffer to:

a. Suspend cells from the agar plates.
b. Wash PFGE plugs after cell lysis.

Cell Suspension Buffer (CSB) - use in E. coli STEC, Salmonella, Shigella spp and Vibrio spp standardized
PulseNet PFGE protocols

100 mM Tris-HCl:100 mM EDTA, pH 8.0

10 ml 1 M Tris-HCl, pH 8.0
20 ml 0.5 M EDTA, pH 8.0
Dilute to 100 ml with sterile Ultrapure H2O

Cell Lysis Buffer - use for lysing E. coli STECs, Salmonella, Shigella spp, Vibrio spp, Listeria
monocytogenes and Campylobacter jejuni cells in PFGE plugs made in reusable (or disposable) plug molds;
plugs are lysed in 50 ml screw-cap tubes.

50 mM Tris-HCl:50 mM EDTA, pH 8.0 + 1% N-Lauroyl-Sarcosine, Sodium salt (Sarcosyl)

0.1 mg/ml Proteinase K (add just before use).

25 ml 1 M Tris-HCl, pH 8.0
50 ml 0.5 M EDTA, pH 8.0

3
This formula for TE is from Molecular Cloning - A Laboratory Manual by J. Sambrook and E. Russell, 3rd edition.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 5
 STANDARD OPERATING PROCEDURE FOR LABORATORY FORMULAS AND CODE: PNL02
Effective Date:
REAGENTS 08 14 2013

50 ml 10% Sarcosyl 3
Dilute to 500 ml with sterile Ultrapure H2O

Add 25 μl Proteinase K stock solution (20 mg/ml) per 5 ml of cell lysis buffer just before use.
The final concentration of Proteinase K is 0.1 mg/ml.
3
Note: These reagents can also be made by adding 5g of Sarcosyl powder directly to the other three
ingredients (Tris-HCl, EDTA, 400 ml H2O). Warm the solution to 50°- 60°C for 30-60 minutes, or leave
at room temperature for 1-2 hours to completely dissolve the Sarcosyl; adjust to the final volume with
sterile Ultrapure H2O.

0.5X TBE Buffer

200 ml 5X TBE Buffer*

Dilute to 2000 ml with Ultrapure H2O
or
100 ml 10X TBE Buffer*
Dilute to 2000 ml with Ultrapure H2O

Note: The water used to dilute concentrated 5X or 10X TBE buffer to 0.5X TBE does not have to be
sterile.

Use of trade names and commercial sources is for identification only and does not imply endorsement by
CDC or the U.S. Department of Health and Human Services.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:
2010-05-07 Updated the Lysozyme Stock Solution and SDS for Listeria monocytogenes to
reflect the changes made to the PFGE protocol PNL04.
2013-08-14 Updated Lysozyme Stock Solution and SDS for Listeria monocytogenes to reflect
the changes made to the PFGE protocol PNL04.
2013-08-14 Clarified language regarding formula for TE.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 5
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

1. PURPOSE: To describe the One-Day (24-26 h) Standardized Laboratory Protocol for Molecular
Subtyping of Campylobacter jejuni by Pulsed-field Gel Electrophoresis (PFGE).

2. SCOPE: To provide the PulseNet participants with a standardized procedure for performing PFGE
of Campylobacter jejuni, thus ensuring inter-laboratory comparability of the generated results.

3. DEFNITIONS / TERMS:
3.1. PFGE: Pulsed-field Gel Electrophoresis
3.2. DNA: Deoxyribonucleic acid
3.3. CDC: Centers for Disease Control and Prevention
3.4. CLRW: Clinical Laboratory Reagent Water

4. RESPONSIBILITIES / PROCEDURE:

BIOSAFETY WARNING: Please read all instructions carefully before starting protocol. Treat all plasticware,
glassware, pipets, spatulas, etc. that come in contact with the cell suspensions or plugs as contaminated materials
and dispose of, or disinfect according to the guidelines of your institution. Disinfect plug molds before they are
washed. Contaminated items should be disinfected with 1% Lysol/Amphyll or 90% ethanol for at least 30 minutes if
they will be washed and reused.

Day 0
Streak an isolated colony from test cultures onto Trypticase Soy Agar with 5% defibrinated sheep blood (TSA-SB)
plates (or comparable non-selective media) for confluent growth. It is recommended that a storage vial of each culture be
created. To do this, stab small screw cap tubes of TSA, HIA, or similar medium with the same inoculating loop used to
streak the plate. This will ensure that the same colony can be retested if necessary. Incubate cultures at microaerobically
37ºC for 14-18 h.

Day 1
1. Turn on shaker water bath or incubator (54-55ºC), stationary water baths (55-60ºC) and
spectrophotometer (or equivalent instrument such as the Dade Microscan Turbidity meter or bioMérieux Vitek
colorimeter).

2. Prepare TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) 1 as follows:

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile, Ultrapure Clinical Laboratory Reagent Water (CLRW)

Note: The TE Buffer used to make the plug agarose is also used to wash lysed PFGE plugs.

3. Prepare 1% SeaKem Gold agarose in TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) as follows:
a. Weigh 0.50 g (or 0.25 g) SeaKem Gold (SKG) into 250 ml screw-cap flask.
b. Add 50.0 ml (or 25.0 ml) TE Buffer; swirl gently to disperse agarose.
c. Loosen cap or cover loosely with clear film and microwave for 30 sec; mix gently and repeat for 10 sec
intervals until agarose is completely dissolved.
d. Recap flask and return to a 55-60ºC water bath and equilibrate the agarose for 15 minutes or until ready to
use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

1
Additional information is found on page 11 of this document.
2
The N-Lauroylsarcosine, Sodium salt can be added directly to the other ingredients and allowed to dissolve. See
page 11 of this document.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 1 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

Note: SeaKem Gold agarose works well for making PFGE plugs because it provides added strength to the plugs
minimizing breakage of plugs during the lysis and washing steps. The time and temperature needed to completely
dissolve the agarose is dependent on the specifications of the microwave used, and will have to be determined
empirically in each laboratory.

4. Label small transparent tubes (12 mm x 75 mm Falcon 2054 tubes or equivalent) with culture numbers.

5. Transfer ~2 ml of phosphate-buffered saline (0.01 M PBS, pH 7.4) or 0.85% NaCl to small labeled tubes
(Falcon 2054 tubes). Use sterile polyester-fiber or cotton swab that has been moistened with sterile PBS to
remove some of the growth from agar plate; suspend cells in PBS by spinning swab gently so cells will be evenly
dispersed and formation of aerosols is minimized.

Note: The minimum volume of the cell suspension needed will depend on size of the cuvettes or tubes used to measure
the cell concentration and are dependent on the manufacturer’s specifications for the spectrophotometer, turbidity meter,
or colorimeter. Keep suspensions on ice if you have more than 6 cultures to process or refrigerate cell suspensions if you
cannot adjust their concentration immediately.

6. Adjust concentration of cell suspensions to one of the values given below by diluting with sterile PBS or by
adding additional cells:
a. Spectrophotometer: 610 nm wavelength, absorbance (Optical Density) of 0.680 (0.570 – 0.820).
b. Dade Microscan Turbidity Meter: 0.35 – 0.45 (measured in Falcon 2054 tubes).
0.52 – 0.64 (measured in Falcon 2057 tubes).
c. bioMérieux Vitek colorimeter: ≈ 20% transmittance (measured in Falcon 2054 tubes)

Note: The values in Steps 6a, 6b and 6c give satisfactory results at CDC; each laboratory may need to establish the
optimal concentration needed for satisfactory results.

CASTING PLUGS

Label wells of PFGE plug molds with culture number. When reusable plug molds are used, put strip of tape on lower
part of reusable plug mold before labeling wells.

Note: Unused plug agarose can be kept at room temperature and reused 1-2 times. Microwave on low-medium power
for 10-15 sec and mix; repeat for 5-10 sec intervals until agarose is completely melted. This agarose melts rapidly!

Note: Proteinase K solutions (20 mg/ml) are available commercially. Alternatively, a stock solution of Proteinase K can
be prepared from the powder in sterile Ultrapure water (CLRW). For best results, aliquot 300-500μl into small tubes and
store in a freezer (-20 ºC) until ready to use. Just before use, thaw appropriate number of vials needed for the samples;
keep Proteinase K solutions on ice. If the Proteinase K stock solution was prepared from powder, discard any thawed
solution at the end of the work day. Store commercially prepared Proteinase K solutions according to directions
provided by the supplier.

1. Transfer 400µl (0.4 ml) of adjusted cell suspensions to labeled 1.5ml microcentrifuge tubes.

2. Add 20 µl of Proteinase K (20 mg/ml stock) to each tube and mix gently with pipet tip (200 µl is needed for 10
cell suspensions).

3. Add 400 µl (0.4 ml) melted 1% SeaKem Gold agarose to 400 µl cell suspension and mix gently by pipeting up
and down two or three times. Over-pipeting can cause DNA shearing. Maintain temperature of melted
agarose by keeping flask in beaker of warm water (55-60ºC).

4. Immediately, dispense part of mixture into appropriate well(s) of disposable plug mold. Do not allow bubbles

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 2 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

to form. Two plugs of each sample can be made from these amounts of cell suspension and agarose and are useful if
repeat testing is required. Allow plugs to solidify at room temperature for 10-15 min. They can also be placed in
the refrigerator (4ºC) for 5 minutes.

Note: If disposable plug molds are used for making plugs with 1% SeaKem Gold agarose, use 200 μl cell suspension,
10 μl of Proteinase K (20 mg/ml stock) and 200 μl of agarose; up to 4 plugs can be made from these amounts of cell
suspension and agarose.

Note: The generation of cell suspension and the subsequent casting of the plugs should be performed as rapidly as
possible in order to minimize premature cell lysis. If large numbers of samples are being prepared, it is recommended
that they be processed in batches of ~10 samples at a time. Once the first batch of isolates are in the cell lysis incubation,
then start preparing the cells suspensions the next group samples, and so on. All batches can be lysed and washed
together, since additional lysis time will not affect the initial batches.

LYSIS OF CELLS IN AGAROSE PLUGS

Note: Two plugs (reusable plug molds) or up to four plugs (disposable plug molds) of the same strain can be lysed
in the same 50 ml tube.

1. Label 50 ml polypropylene screw-cap or 50 ml Oak Ridge tubes with culture numbers.

2. Prepare Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) as follows:
25 ml of 1 M Tris, pH 8.0
50 ml of 0.5 M EDTA, pH 8.0
50 ml of 10 % Sarcosyl (N-Lauroylsarcosine, Sodium salt) 2
Dilute to 500 ml with sterile Ultrapure water (CLRW)

3. Calculate the total volume of Cell Lysis/Proteinase K Buffer needed as follows:

a. 5 ml Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) is needed per tube
(e. g., 5 ml x 10 tubes = 50 ml).
b. 25 µl Proteinase K stock solution (20 mg/ml) is needed per tube of the cell lysis buffer
(e. g., 25 µl x 10 tubes = 250 µl).
c. Prepare the master mix by measuring the correct volume of Cell Lysis Buffer and Proteinase K into appropriate
size test tube or flask and mix well.

Note: The final concentration of Proteinase K in lysis buffer is 0.1 mg/ml and is different from the concentration
that was added to the cell suspension (0.5 mg/ml).

4. Add 5 ml of Proteinase K/Cell Lysis Buffer to each labeled 50 ml tube.

5. Trim excess agarose from top of plugs with scalpel, razor blade or similar instrument. Open reusable plug mold
and transfer plugs from mold with a 6 mm wide spatula to appropriately labeled tube. If disposable plug molds are
used, remove white tape from bottom of mold and push out plug(s) into appropriately labeled tube. Be sure plugs
are under buffer and not on side of tube.

Note: The excess agarose, scalpel, spatula, tape, etc. are contaminated. Dispose of or disinfect them appropriately.

6. Remove tape from reusable mold. Place both sections of plug mold, spatulas, and scalpel in 90% ethanol, 1%
Lysol/Amphyll or other suitable disinfectant. Soak them for 15 minutes before washing them. Discard

2
The N-Lauroylsarcosine, Sodium salt can be added directly to the other ingredients and allowed to dissolve. See
page 11 of this document.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 3 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

disposable plug molds.

7. Place tubes in rack and incubate in a 54-55ºC shaker water bath for 15-30 min with constant and vigorous
agitation (175-200 rpm). If lysing in water bath, be sure water level in water bath is above level of lysis buffer
in tubes.

8. Pre-heat enough sterile Ultrapure water (CLRW) to 54-55ºC so that plugs can be washed two times with 10-15
ml water (200-250 ml for 10 tubes).

WASHING OF AGAROSE PLUGS AFTER CELL LYSIS

Note: Most laboratories will find that their plugs are sufficiently stable to perform the following washing steps at
54-55ºC. However, if you notice that your plugs are nicked along the edges or breaking it will be necessary for your
laboratory to lower the water bath or incubator to 50ºC for the following washing steps.

1. Remove tubes from water bath and carefully pour off lysis buffer. Plugs can be held in tubes with a screened
cap (Bio-Rad) or spatula.

Note: Be sure to remove all of the liquid during this and subsequent wash steps by touching lip of tube onto an
absorbent paper towel.

2. Add 10-15 ml of sterile Ultrapure water (CLRW) that has been pre-heated to 54-55°C to each tube and shake the
tubes vigorously in a 54-55°C water bath or incubator for 10-15 min.

3. Pour off water from the plug and repeat wash step with pre-heated water (Step 2) one more time.
a. Pre-heat enough sterile TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) in a 54-55°C water bath so that
plugs can be washed 4 times with 10-15 ml TE (400-600 ml for 10 tubes) after beginning last water wash.

4. Pour off water, add 10-15 ml pre-heated (54-55°C) sterile TE Buffer, and shake the tubes vigorously in 54-55°C
incubator or water bath for 10-15 min.

5. Pour off TE and repeat wash step with pre-heated TE three more times.

6. Decant last wash and add 5-10 ml sterile TE. Continue with step 1 in "Restriction Digestion" section or store plugs
in TE Buffer at 4ºC until needed. Plugs can be transferred to smaller tubes for storage.

Note: If restriction digestion is to be done the same day, complete Steps 1-3 of next section (Restriction Digestion)
during last TE wash step for optimal use of time.

RESTRICTION DIGESTION OF DNA IN AGAROSE PLUGS

Note: A small slice of the plug (not the entire plug) should be digested with the primary restriction enzyme SmaI
because less enzyme is required and other slices of the plug can be subjected to restriction analysis with other
enzymes. KpnI is recommended as the secondary enzyme for analysis of Campylobacter jejuni isolates. The use of
a secondary enzyme is useful in situations where the PFGE patterns obtained with the primary enzyme from two or
more isolates are indistinguishable

1. Label 1.5 ml microcentrifuge tubes with culture numbers; label 3 (10-well gel) or 4 (15-well gel) tubes for
Salmonella ser. Braenderup H9812 3 standards.

3
Directions for making and testing PFGE plugs of Salmonella ser. Braenderup H9812 are in PNL05.
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 4 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

a. Pre-Restriction Incubation Step (highly-recommended): Prepare a master mix by diluting the appropriate
10X restriction buffer (Roche Applied Science or equivalent) 1:10 with sterile Ultrapure water (CLRW)
according to the following table:

Note: The appropriate restriction buffer will vary between vendors and may differ between enzymes from the same
vendor. Always use the restriction buffer recommended by the vendor for the particular restriction enzyme.

Reagent µl/Plug Slice µl/10 Plug Slices µl/15 Plug Slices

Sterile Clinical Laboratory 180 µl 1800 µl 2700 µl
Reagent Water (CLRW)
10X Restriction Buffer 20 µl 200 µl 300 µl
Total Volume 200 µl 2000 µl 3000 µl

b. Add 200 µl diluted restriction buffer (1X) to labeled 1.5 ml microcentrifuge tubes.
c. Carefully remove plug from TE with spatula and place in a sterile disposable Petri dish or on large glass
slide.
d. Cut a 2.0 to 2.5 mm wide slice from each test sample and the appropriate number of S. ser. Braenderup
H9812 standards with a scalpel (or single edge razor blade, cover slip, etc.) and transfer to tube containing
diluted restriction buffer. Be sure plug slice is under buffer. Replace rest of plug in original tube that
contains 5 ml TE buffer and store at 4°C.

Note: PulseNet recommends that the combs with larger teeth (10 mm wide teeth) be used to cast the gels because
computer analysis of the gel lanes is more accurate and less tedious than analysis of gel lanes cast with combs with the
smaller teeth (5.5 mm). Using combs with smaller teeth is not advised. The number of slices that can be cut from the
plugs will depend on the skill and experience of the operator, integrity of the plug, and whether the slices are cut
vertically or horizontally (plugs made in disposable molds).

e. Incubate sample and control plug slices in water bath or incubator for 5-10 min or at room temperature for
10-15 min.
i. Incubate samples to be restricted with SmaI at 25°C
ii. Incubate samples to be restricted with KpnI and XbaI at 37°C.
f. After incubation, remove buffer from plug slice using a pipet fitted with 200-250 µl tip all the way to
bottom of tube and aspirate buffer. Be careful not to cut plug slice with pipet tip and that plug slice is not
discarded with pipet tip.

2. Prepare the restriction enzyme master mix according to the following table. May mix in the same tube that was
used for the diluted restriction buffer:

Note: Enzymes may be purchased in several different stock concentrations. The calculations below are based on using
an enzyme at a concentration of 40 U/μl. If a different concentration of enzyme is used, make necessary adjustments to
the volume of enzyme and water to achieve a final concentration of 40 U/ sample.

Note: Keep vial of restriction enzyme on ice or in insulated storage box (-20°C) at all times.

Reagent µl/Plug Slice µl/10 Plug Slices µl/15 Plug Slices

Sterile Clinical Laboratory
Reagent Water (CLRW) 177 µl 1770 µl 2655 µl

10X Restriction Buffer 20 µl 200 µl 300 µl

BSA (10mg/ml) 2 µl 20 µl 30 µl
SmaI (40 U/µl) 1 µl 10 µl 15 µl

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 5 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

Total Volume 200 µl 2000 µl 3000 µl

Reagent µl/Plug Slice µl/10 Plug Slices µl/15 Plug Slices

Sterile Clinical Laboratory
Reagent Water (CLRW) 177 µl 1770 µl 2655 µl

10X Restriction Buffer 20 µl 200 µl 300 µl

BSA (10mg/ml) 2 µl 20 µl 30 µl
KpnI (40 U/µl) 1 µl 10 µl 15 µl
Total Volume 200 µl 2000 µl 3000 µl

Note: Addition of Bovine Serum Albumin (BSA; highly recommended): Several restriction enzyme vendors
specifically recommend the addition of 1X BSA to enzyme restriction mixtures while others do not. PulseNet
Central recommends adding BSA to all enzyme restriction mixtures to minimize the incidence of incomplete
restriction.

3. Add 200 µl restriction enzyme master mix to each tube. Close tube and mix by tapping gently; be sure plug
slices are under enzyme mixture.

4. Incubate sample and standard (control) plug slices for 2 hours (unless indicated otherwise) in a water bath or
incubator at the appropriate temperature for the enzyme.
a. Incubate samples restricted with SmaI at 25°C.
b. Incubate samples restricted with KpnI (4 – 6 hours) and XbaI at 37°C.

5. If plug slices will be loaded into the wells (Option B, page 7), continue with Steps 1-4 of the next section
(CASTING AGAROSE GEL) approximately 1 h before restriction digest reaction is finished so the gel can
solidify for at least 30 minutes before loading the restricted PFGE plugs.

CASTING AGAROSE GEL

A. Loading Restricted Plug Slices on the Comb:

1. Confirm that water bath is equilibrated to 55-60°C.

2. Make volume of 0.5X Tris-Borate EDTA Buffer (TBE) that is needed for both the gel and electrophoresis
running buffer according to one of the following tables.

5X TBE:
Reagent Volume in milliliters (ml)
5X TBE 200 220
Clinical Laboratory Reagent
1800 1980
Water (CLRW)
Total Volume of 0.5X TBE 2000 2200

10X TBE:
Reagent Volume in milliliters (ml)

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 6 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

10X TBE 100 110

Clinical Laboratory Reagent
1900 2090
Water (CLRW)
Total Volume of 0.5X TBE 2000 2200

3. Make 1% SeaKem Gold (SKG) Agarose in 0.5X TBE as follows:

a. Weigh appropriate amount of SKG into 500 ml screw-cap flask.
b. Add appropriate amount of 0.5X TBE; swirl gently to disperse agarose.
i. Mix 1.0 g agarose with 100 ml 0.5X TBE for 14 cm wide gel form (10wells)
ii. Mix 1.5 g agarose with 150 ml 0.5X TBE for 21 cm wide gel form (15 wells)
c. Loosen cap or cover loosely with clear film and microwave for 60 sec; mix gently and repeat for 15 sec
intervals until agarose is completely dissolved.
d. Recap flask and return to 55-60ºC water bath and equilibrate the agarose in the water bath for 15 minutes or
until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: Agarose LFTM (Amresco, X174) is the only acceptable alternative to SeaKem Gold, at this time. The time and
temperature needed to completely dissolve the agarose is dependent on the specifications of the microwave used and will
have to be determined empirically in each laboratory. Similarly, the optimal running time for each agarose will have to
be determined empirically in each laboratory.

4. A small volume (2-5 ml) of melted and cooled (50-60ºC) 1% SKG agarose may be added to fill wells after plugs
are loaded. Prepare as described above. Unused SKG agarose can be kept at room temperature, melted, and
reused several times.

Note: Place the gel form on a leveling table and adjust until perfectly leveled. Place the comb holder so the front
part (side with small metal screws) and teeth face the bottom of gel frame and the comb teeth touch the gel platform.

5. Remove restricted plug slices from water bath. Remove enzyme/buffer mixture and add 200 µl 0.5X TBE.
Incubate at room temperature for 5 min.

6. Remove plug slices from tubes; put comb on bench top and load plug slices on the bottom edge of the teeth in
the following order:
a. Load Salmonella serotype Branderup H9812 standards in lanes (teeth) 1, 5, 10 (10 well gel) or in lanes 1, 5,
10, 15 (15 well gel).
b. Load samples on remaining teeth of the comb and note locations.

7. Remove excess buffer with tissue or kimwipe. Allow plug slices to air dry on the comb for 5-10 minutes or seal
them to the comb with 1% SKG agarose (55-60°C).

8. Position comb in leveled gel form and confirm that the plugs slices are correctly aligned on the bottom of the
comb teeth and that the lower edge of the plug slice is flush against the black platform.

9. Carefully pour the agarose (cooled to 55-60ºC) into the gel form and remove any bubbles or debris.

10. Put black gel frame in electrophoresis chamber. Add 2-2.2 L freshly prepared 0.5X TBE. Close cover of unit.
The amount of buffer needed depends on whether residual buffer was left in tubing or if unit was flushed with
water after the last gel was run.

11. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute (setting of ≈70) and cooling module (14ºC).

12. Remove comb after gel solidifies, about 30-45 minutes.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 7 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

13. Fill in wells of gel with melted and cooled (55-60ºC) 1% SKG Agarose (optional). Unscrew and remove end
gates from gel form; remove excess agarose from sides and bottom of casting platform with a tissue or
kimwipe. Keep gel on casting platform and carefully place gel inside black gel frame in electrophoresis
chamber. Close cover of chamber.

B. Loading Restricted Plug Slices into the Wells:

1. Follow steps 1-4 in “Option A” above (Loading Restricted Plug Slices on the Comb).

Note: Place the gel form on a leveling table and adjust until perfectly leveled before pouring gel. Position the comb
holder so that front part (side with small metal screws) and teeth face the bottom of the gel and the bottom of the
comb is 2mm above the surface of the gel platform.

2. Cool melted SKG agarose in 55-60°C water bath for 15-20 min; carefully pour agarose into gel form (casting
stand) fitted with comb. Be sure there are no bubbles.

3. Put black gel frame in electrophoresis chamber. Add 2-2.2 L freshly prepared 0.5X TBE. Close cover of unit.
The amount of buffer depends on whether residual buffer was left in tubing, or if unit was flushed with water
after the last gel was run.

4. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute and cooling module (14°C)
approximately 30 minutes before gel is to be run.

5. Remove restricted plug slices from water bath. Remove enzyme/buffer mixture and add 200 µl 0.5X TBE.
Incubate at room temperature for 5 minutes.

6. Remove comb after gel solidifies, about30 – 45 minutes.

7. Remove restricted plug slices from tubes with tapered end of spatula and load into appropriate wells. Gently
push plugs to bottom and front of wells with wide end of spatula. Manipulate position with spatula and be sure
that are no bubbles.
a. Load Salmonella serotype Branderup H9812 standards in lanes 1, 5, 10 (10-well gel) or in lanes 1, 5, 10, 15
(15-well gel).
b. Load samples in remaining wells.

Note: Loading the plug slices can be tedious; each person has to develop his/her own technique for consistently
placing the plug slices in the wells so the lanes will be straight and the bands sharp.

8. Fill in wells of gel with melted 1% SKG Agarose (equilibrated to 55-60°C). Allow to harden for 3-5 min.
Unscrew and remove end gates from gel form; remove excess agarose from sides and bottom of casting
platform with a tissue or kimwipe. Keep gel on casting platform and carefully place gel inside black gel frame
in electrophoresis chamber. Close cover of chamber.

ELECTROPHORESIS CONDITIONS

1. Select following conditions for Campylobacter jejuni restricted with SmaI.

a. Select following conditions on Chef Mapper

Auto Algorithm
50 kb - low MW
400 kb - high MW
Select default values except where noted by pressing "enter"
Change run time to 18 – 19 hours (see note below)

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 8 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

(Default values: Initial switch time = 6.76 s; Final switch time = 35.38 s)

b. Select following conditions on CHEF-DR III

Initial switch time: 6.8 s
Final switch time: 35.4 s
Voltage: 6 V
Included Angle: 120°
Run time: 18 – 19 h (see note below)

c. Select following conditions on CHEF-DR II

Initial A time: 6.8 s
Final A time: 35.4 s
Start ratio: 1.0 (if applicable)
Voltage: 200 V
Run time: 19 – 20 h (see note below)

2. Select following conditions on Chef Mapper for Campylobacter jejuni restricted with KpnI.

a. Select following conditions on Chef Mapper

Auto Algorithm
50 kb - low MW
475 kb - high MW
Select default values except where noted by pressing "enter"
Change run time to 18 – 19 hours (see note below)
Change initial switch time = 5.2 s. Accept default Final switch time = 42.34 s.

b. Select following conditions on CHEF-DR III

Initial switch time = 5.2 s
Final Switch time = 42.3 s
Voltage: 6 V
Included Angle: 120°
Run time: 18 – 19 h (see note below)

c. Select following conditions on CHEF-DR II

Initial A time: 5.2 s
Final A time: 42.3 s
Start ratio: 1.0 (if applicable)
Voltage: 200 V
Run time: 19 – 20 h (see note below)

Note: The electrophoresis running times recommended above are based on the equipment and reagents used at the
CDC. Run times may be different in your laboratory and will have to be optimized for your gels so that the
lowest band in the S. ser. Braenderup H9812 standard migrates 1.0 - 1.5 cm from the bottom of the gel.

Note: Make note of the initial milliamp (mAmp) reading on the instrument. The initial mAmps should be between 110-
150 mAmps. A reading outside of this range may indicate that the 0.5X TBE buffer was prepared improperly and the
buffer should be remade.

Day 2

STAINING AND DOCUMENTATION OF PFGE AGAROSE GEL

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 9 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

Note: The following staining procedure describes the use of ethidium bromide to stain PFGE gels. Alternate DNA
stains may be used. Please see the “Alternate DNA Stains-Results and Recommendations” posting within the PulseNet
Documents forum on the SharePoint site for additional information.

1. When electrophoresis run is over, turn off equipment; remove and stain gel with ethidium bromide by diluting
40 µl of ethidium bromide stock solution (10 mg/ml) with 400 ml of reagent grade water. This volume is for a
staining box that is approximately 14 cm x 24 cm; a larger container may require a larger amount of staining
solution. Stain gel for 20-30 min in covered container.

Note: Ethidium bromide is toxic and a mutagen. Stock solutions of 10 mg/ml Ethidium Bromide (EtBr) in water are
available from several commercial companies (Amresco X328; Bio-Rad, 161-0433; Sigma, E-1510). The diluted
solution can be kept in dark bottle and reused 6-8 times before discarding according to your institution's guidelines
for hazardous waste. CDC does not recommend disposing of EtBr down the drain. Aqueous solutions containing
EtBr can be filtered through charcoal or degraded using activated carbon destaining or “tea” bags from Amresco
(E732-25 Destaining Bags) or other companies, which effectively and safely remove EtBr from solutions and gels.
Once the EtBr is removed, the treated aqueous solutions can be discarded down the drain. If you have further
questions about EtBr please refer to the Material Safety Data Sheets (MSDS) provided by the vendor or
manufacturer.

Note: Currently, the only acceptable alternative stain options are GelRedTM (Biotium, 31010), SYBR® Safe
(Invitrogen, S-33102) and SYBR® Gold (Invitrogen, S-11494). Labs are strongly encouraged to follow
manufacturer’s instructions and test stains in their labs before adopting them for routine use. If one of the
alternative stains is used, the destaining steps should be omitted.

2. Destain gel in approximately 500 ml CLRW for 60-90 min, changing water every 20 minutes. Capture image on
Gel Doc 1000, 2000, EQ, XR, or equivalent documentation system. If background interferes with resolution,
destain for an additional 30-60 min.

3. Follow directions given with the imaging equipment to save gel image as an *.1sc file; convert this file to *.tif
file for analysis with BioNumerics software program. The gel image should fill the entire window of the
imaging equipment (computer) screen (without cutting off wells or lower bands). Ensure that the image is in
focus and that there is little to no saturation (over-exposure) in the bands (signified by red pixilation in the
QuantityOne or ImageLab software). Additional instructions are provided in PNL07 of the PulseNet QA/QC
manual.

4. Drain buffer from electrophoresis chamber and discard. Rinse chamber with 2 L CLRW or, if unit is not going
to be used for several days, flush lines with water by letting pump run for 5-10 min before draining water from
chamber and tubing.

5. If the lowest band in the H9812 standard does not migrate within 1-1.5 cm of the bottom of the gel, the proper run
time will need to be determined empirically for the conditions in each laboratory.

Note: The following options are available if PFGE results do not have to be available within 24 hours:
− Plugs can be lysed for longer periods of time (up to 2 hours).
− The washing steps with TE to remove the lysis buffer from the PFGE plugs can be done for longer
periods of time (15-30 min) and at lower temperatures (37ºC or room temperature). They can be
started on Day 1 and finished the morning of Day 2 after overnight refrigeration of the plugs in TE.

Use of trade names and commercial sources is for identification purposes only and does not imply endorsement by
CDC or the U.S. Department of Health and Human Services.

NOTE: CLIA LABORATORY PROCEDURE MANUAL REQUIREMENTS

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 10 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

Efforts have been made to assure that the procedures described in this protocol have been written in accordance with
the 1988 Clinical Laboratory Improvement Amendments (CLIA) requirements for a procedure manual (42 CFR
493.1211). However, due to the format required for training, the procedures will require some modifications and
additions to customize them for your particular laboratory operation.
Any questions regarding the CLIA requirements for a procedure manual, quality control, quality assurance, etc.,
should be directed to the agency or accreditation organization responsible for performing your laboratory's CLIA
inspection. In addition, some states and accreditation organizations may have more stringent requirements that will need
to be addressed.

Formulas of Selected Reagents used in PulseNet Standardized Laboratory Protocol for PFGE

Tris:EDTA Buffer, pH 8.0 (TE, 10 mM Tris:1 mM EDTA, pH 8.0)

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure water (CLRW)

Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosine + 0.1 mg/ml Proteinase K)
25 ml (50 ml) of 1 M Tris, pH 8.0
50 ml (100 ml) of 0.5 M EDTA, pH 8.0
50 ml (100 ml) 10% N-Lauroylsarcosine, Sodium salt (Sarcosyl)
OR
5 g (10 g) of N-Lauroylsarcosine, Sodium salt (Sarcosyl) 4
Dilute to 500 ml (1000 ml) with Sterile Ultrapure water (CLRW)

Add 25 μl Proteinase K stock solution (20 mg/ml) per 5 ml of cell lysis buffer just before use for a final
concentration in the lysis buffer of 0.1 mg/ml Proteinase K.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:
8.1 The phrase “Type I Water” has been changed to “Ultrapure Clinical Laboratory Reagent Water (CLRW).”
The water composition is the same, but this reflects a change in the terminology used by the Clinical
Laboratory Standards Institute (CLSI).
8.2 2011-08 changes:
− The wording for programming electrophoresis conditions was updated to standardize this section and make
it the same as other PFGE laboratory SOPs.
− The wording for washing the agarose plugs after cell lysis was also updated to standardize the section.
8.3 2013-03 changes:
− Corrected formula for TE buffer. TE used at CDC is 10 mM for Tris and 1 mM for EDTA.
− Recommended disinfectant changed from 10% bleach to 1% Lysol/Amphyll or 90% ethanol.
− Volume of TE needed for washing plugs was correted from 300 – 350 ml to 400 – 600 ml.
− A statement was added to clarify that using combs with small teeth (5.5 mm) was not advised.
− Use of pre-restriction step and BSA was changed from optional to highly recommended. Calculation
for including BSA in restriction enzyme master mix was added.

4
If Sarcosyl powder is added directly to the other components of this reagent, warm the solution to 50- 60ºC for 30-
60 minutes, or leave at room temperature for ≈2 hours to completely dissolve the Sarcosyl; adjust to the final
volume with sterile Ultrapure water (CLRW).
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 11 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CODE: PNL03
Effective Date:
CAMPYLOBACTER JEJUNI 03 24 2013

− Statement allowing of Megbase agarose (BioRad) was deleted. Additional testing revealed run time
and normalization were negatively impacted by this agarose.
− A statement was included to allow the use of alternative DNA stains that are equivalent to EtBr. Labs
are strongly urged to follow manufacturer’s instructions as well as test stains in their own labs to gain
experience using alternative agarose stains. Additional stain alternatives may be tested and deemed
acceptable at a later date.
− The option to allow incubation times for restriction digestion to be increased longer than recommended
was deleted.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 12 of 12
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

1. PURPOSE: To describe the One-Day (24-26 h) standardized laboratory protocol for molecular
subtyping of Listeria monocytogenes by Pulsed-field Gel Electrophoresis (PFGE).

2. SCOPE: To provide the PulseNet participants with a standardized procedure for performing PFGE
of Listeria monocytogenes thus ensuring inter-laboratory comparability of the generated results.

3. DEFINITIONS/TERMS:
3.1 PFGE: Pulsed-field Gel Electrophoresis
3.2 DNA: Deoxyribonucleic acid
3.3 CDC: Centers for Disease Control and Prevention
3.4 CLRW: Clinical Laboratory Reagent Water

4. RESPONSIBILITIES/PROCEDURE:

PREPARATION OF PFGE PLUGS FROM AGAR CULTURES

BIOSAFETY WARNING: The infectious dose for listeriosis has not been determined and it may depend, in part,
on the susceptibility of the host. Groups at highest risk of acquiring infection are pregnant women, neonates,
immunocompromised patients, and the elderly. Therefore, laboratorians working with Listeria monocytogenes,
particularly those who may be at increased risk of acquiring listeriosis should be made aware of this potential and
advised to be particularly cautious when working with this organism.

Please read all instructions carefully before starting protocol. Treat all plasticware, glassware, pipets, spatulas, etc. that
come in contact with the cell suspensions or plugs as contaminated materials and dispose of, or disinfect according to the
guidelines of your institution. Disinfect reusable plug molds before they are washed; the disposable plug molds,
including the tape and the tab that is used to push the plugs out of the wells, are also contaminated and should be
disinfected with 1% Lysol/Amphyll or 90% ethanol for at least 30 minutes if they will be washed and reused.

Day 0
Streak an isolated colony from test cultures onto Brain Heart Infusion Agar (BHIA) plates (or comparable non-selective
media) for confluent growth. It is recommended that a storage vial of each culture be created. To do this, stab small
screw cap tubes of TSA, HIA, or similar medium with the same inoculating loop used to streak the plate. This will
ensure that the same colony can be retested if necessary. Incubate cultures at 37ºC for 14-18 h.

Day 1
1. Turn on shaker water bath or incubator (54-55ºC), stationary water baths (55-60ºC) and spectrophotometer
(or equivalent instrument such as the Dade Microscan Turbidity meter or bioMérieux Vitek colorimeter).

2. Prepare TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0)1 as follows:

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure water (Clinical Laboratory Reagent Water (CLRW))

Note: The TE Buffer is used to make the plug agarose, suspend the cells, and to wash lysed PFGE plugs.

3. Prepare Lysozyme stock solution as follows:

1
Additional information is found on page 12 of this document.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 1 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

a. For 20mg/ml stock, weigh out 100 mg Lysozyme (Sigma L7651 or L6875). Keep the container of Lysozyme
on ice.
b. Add 5 mL TE buffer, swirl to mix,
c. Aliquot 250 µL amounts into sterile eppendorf tubes and freeze for future use.
d. Or for 10mg/ml stock, weight out 50 mg Lysozyme (Sigma L1667). Keep the container of Lysozyme on ice.
e. Add 5 mL TE buffer, swirl to mix.
f. Aliquot 250 µL amounts into sterile eppendorf tubes and freeze for future use.

4. Prepare 1% SeaKem Gold agarose + 0.5% Sodium Dodecyl Sulfate (SDS) in TE Buffer (10 mM Tris:1 mM
EDTA, pH 8.0) for PFGE plugs as follows:
a. Place 10% SDS stock solution (GibcoBRL #15553-035) into 55- 60ºC water bath to warm.
b. Weigh 0.50 g (or 0.25 g) SeaKem Gold (SKG) agarose into 250 ml screw-cap flask.
c. Add 47.5 ml (or 23.75 ml) TE Buffer; swirl gently to disperse agarose.
d. Loosen or remove cap and cover loosely with clear film, and microwave for 30 sec; mix gently and repeat for
10 sec intervals until agarose is completely dissolved.
e. Add 2.5 mL (or 1.25 mL) of warm 10% SDS stock solution, swirl to mix.
f. Recap flask and return to 55- 60ºC water bath and equilibrate the agarose in the water bath for 15 minutes or
until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: SeaKem Gold agarose works well for making PFGE plugs because it provides added strength to the plugs
minimizing breakage of plugs during the lysis and washing steps. The time and temperature needed to completely
dissolve the agarose is dependent on the specifications of the microwave used, and will have to be determined
empirically in each laboratory.

5. Label small transparent tubes (12 mm x 75 mm Falcon 2054 tubes or equivalent) with culture numbers.

6. Transfer ≈2 ml of TE Buffer to small labeled tubes. Use a sterile polyester-fiber or cotton swab that has been
moistened with sterile TE to remove some of the growth from the agar plate; suspend cells in TE by spinning swab
gently so cells will be evenly dispersed and formation of aerosols is minimized.

Note: The minimum volume of the cell suspension needed will depend on size of the cuvettes or tubes used to measure
the cell concentration and are dependent on the manufacturer’s specifications for the spectrophotometer, turbidity meter,
or colorimeter. Keep suspensions on ice if you have more than 6 cultures to process at one time.

7. Adjust concentration of cell suspensions to one of values given below by diluting with sterile TE or by adding
additional cells.
a. Spectrophotometer: 610 nm wavelength, absorbance (Optical Density) of 1.00
b. Dade Microscan Turbidity Meter: 0.40 - 0.45 (measured in Falcon 2054 tubes)
0.58 - 0.63 (measured in Falcon 2057 tubes)
c. bioMérieux Vitek colorimeter: ≈17-18% transmittance (measured in Falcon 2054 tubes)

Note: Cell suspensions need to be at room temperature when concentration is checked. The values in Steps 7a, 7b and 7c
give satisfactory results at CDC; if different instruments or tubes are used, each laboratory may need to establish the
concentration needed for satisfactory results.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 2 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

CASTING PLUGS

Label wells of PFGE plug molds with culture number. When reusable plug molds are used, put strip of tape on lower
part of reusable plug mold before labeling wells.

Note: Unused plug agarose can be kept at room temperature and reused 1-2 times. Microwave on low-medium power
for 10 -15 sec and mix; repeat for 5-10 sec intervals until agarose is completely melted. This agarose melts rapidly!

Note: Proteinase K solutions (20 mg/ml) are available commercially. Alternatively, a stock solution of Proteinase K can
be prepared from the powder in sterile Ultrapure water (CLRW). For best results, aliquot 300-500 μl into small tubes
and store in a freezer (-20ºC) until ready to use. Just before use, thaw appropriate number of vials needed for the
samples; keep Proteinase K solutions on ice. If the Proteinase K stock solution was prepared from powder, discard any
thawed solution at the end of the work day. Store commercially prepared Proteinase K solutions according to directions
provided by the supplier.

1. Transfer 400 μl (0.4 ml) adjusted cell suspensions to labeled 1.5 ml microcentrifuge tubes.

2. Add 20 μL thawed Lysozyme stock solution (20 mg/mL or 10 mg/mL 2) to each tube and mix gently. Place tubes
into a 55-60ºC water bath for 10-20 minutes. Discard unused thawed Lysozyme solution.

3. Add 20 μl of Proteinase K (20 mg/ml stock) to each tube and mix gently with pipet tip. (200 μl is needed for 10 cell
suspensions.)

4. Add 400 μl (0.4 ml) melted 1% SeaKem Gold agarose to 400 µl cell suspension and mix by gently pipetting
mixture up and down a few times. Over-pipeting can cause DNA shearing. Maintain temperature of melted
agarose by keeping flask in beaker of warm water (55-60ºC).

5. Immediately, dispense part of mixture into appropriate well(s) of reusable plug mold. Do not allow bubbles to form.
Two plugs of each sample can be made from these amounts of cell suspension and agarose. Allow plugs to solidify
at room temperature for 10-15 min. They can also be placed in the refrigerator (4ºC) for 5 minutes.

Note: If disposable plug molds are used for making plugs with 1% SeaKem Gold agarose, use 200 μl cell suspension,
10 μl of Lysozyme (20 mg/ml),10 μl of Proteinase K (20 mg/ml stock), and 200 μl of agarose; up to 4 plugs can be made
from these amounts of cell suspension and agarose.

Note: The generation of cell suspension and the subsequent casting of the plugs should be performed as rapidly as
possible in order to minimize premature cell lysis. If large numbers of samples are being prepared, it is recommended
that they be processed in batches of ~10 samples at a time. Once the first batch of isolates are in the cell lysis incubation,
then start preparing the cells suspensions the next group samples, and so on. All batches can be lysed and washed
together, since additional lysis time will not affect the initial batches.

LYSIS OF CELLS IN AGAROSE PLUGS

Note: Two plugs (reusable plug molds) or up to four plugs (disposable plug molds) of the same strain can be lysed
in the same 50 ml tube.

1. Label 50 ml polypropylene screw-cap or 50 ml Oak Ridge tubes with culture numbers.

2
The same volume is added of either stock solution because L1667 has approximately twice the activity of L6875 or
L7651, according to Sigma.
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:
Page 3 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

2. Prepare Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) as follows:
25 ml of 1 M Tris, pH 8.0
50 ml of 0.5 M EDTA, pH 8.0
50 ml of 10 % Sarcosyl (N-Lauroylsarcosine, Sodium salt) 3
Dilute to 500 ml with sterile Ultrapure water (CLRW)

3. Calculate the total volume of Cell Lysis/Proteinase K Buffer needed as follows:

a. 5 ml Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) is needed per tube
(e. g., 5 ml x 10 tubes = 50 ml).
b. 25 μl Proteinase K stock solution (20 mg/ml) is needed per tube of the cell lysis buffer
(e. g., 25 μl x 10 tubes = 250 μl).
c. Prepare the master mix by measuring the correct volume of Cell Lysis Buffer and Proteinase K into appropriate
size test tube or flask and mix well.

Note: The final concentration of Proteinase K in the lysis buffer is 0.1 mg/ml and is different from the concentration that
was added to the cell suspension (0.5 mg/ml).

4. Add 5 ml of Proteinase K/Cell Lysis Buffer to each labeled 50 ml tube.

5. Trim excess agarose from top of plugs with scalpel, razor blade or similar instrument. Open reusable plug mold and
transfer plugs from mold with a 6 mm wide spatula to appropriately labeled tube. If disposable plug molds are used,
remove white tape from the bottom of mold and push out plug(s) into appropriately labeled tube. Be sure plugs are
under buffer and not on side of tube.

Note: The excess agarose, plug mold, spatula, etc. are contaminated. Discard or disinfect appropriately.

6. Remove tape from reusable mold. Place both sections of the plug mold, spatulas, and scalpel in 90% ethanol, 1%
Lysol/Amphyll or other suitable disinfectant. Soak them for 15 minutes before washing them. Discard
disposable plug molds.

7. Place tubes in rack and incubate in a 54-55ºC shaker water bath or incubator for 2 h with constant and vigorous
agitation (150-175 rpm). If lysing in water bath, be sure water level is above level of lysis buffer in tubes.

8. Pre-heat enough sterile Ultrapure water (CLRW) to 54-55ºC so that plugs can be washed two times with 10-15 ml
water (200-250 ml for 10 tubes).

WASHING OF AGAROSE PLUGS AFTER CELL LYSIS

Note: Most laboratories will find that their plugs are sufficiently stable to perform the following washing steps at
54-55ºC. However, if you notice that your plugs are nicked along the edges or breaking it will be necessary for your
laboratory to lower the water bath or incubator to 50ºC for the following washing steps.

1. Remove tubes from water bath or incubator, and carefully pour off lysis buffer into an appropriate discard container;
plugs can be held in tubes with a screened cap or spatula.

3
The N-Lauroylsarcosine, Sodium salt can be added directly to the other ingredients and allowed to dissolve. See
page 12 of this document.
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:
Page 4 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

Note: It is important to remove all of the liquid during this and subsequent wash steps by touching edge of tube or
screened cap on an absorbent paper towel.

2. Add at 10-15 ml sterile Ultrapure water (CLRW) that has been pre-heated to 54-55ºC to each tube and shake the
tubes in a 54-55ºC water bath or incubator for 10-15 min.

3. Pour off water from the plugs and repeat wash step with pre-heated water (Step 2) one more time.
a. Pre-heat enough sterile TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) in a 54-55ºC water bath so that plugs
can be washed four times with 10-15 ml TE (400-600 ml for 10 tubes) after beginning last water wash.

4. Pour off water, add 10-15 ml pre-heated (54-55ºC) sterile TE Buffer, and shake the tubes in 54-55ºC water bath or
incubator for 10-15 min.

5. Pour off TE and repeat wash step with pre-heated TE three more times.

6. Decant last wash and add 5-10 ml sterile TE. Continue with step 1 in "Restriction Digestion" section or store plugs
in TE Buffer at 4ºC until needed. Plugs can be transferred to smaller tubes for storage.

Note: If restriction digestion is to be done the same day, complete Steps 1-3 of next section (RESTRICTION
DIGESTION OF DNA IN AGAROSE PLUGS WITH AscI or ApaI) during last TE wash step for optimal use of
time.

RESTRICTION DIGESTION OF DNA IN AGAROSE PLUGS

Note: A small slice of the plug (not the entire plug) should be digested with the primary restriction enzyme AscI because
less enzyme is required and other slices of the plug can be subjected to restriction analysis with other enzymes. ApaI is
recommended as the secondary enzyme for analysis of Listeria monocytogenes isolates. The use of a secondary enzyme
is useful in situations where the PFGE patterns obtained with the primary enzyme are also indistinguishable.

1. Label 1.5 ml microcentrifuge tubes with culture numbers; label 3 (10-well gel) or 4 (15-well gel) tubes for
Salmonella ser. Braenderup H9812 4 standards.
a. Pre-Restriction Incubation Step (highly recommended): Prepare a master mix by diluting the appropriate
10X restriction buffer (Roche Applied Science or equivalent) 1:10 with sterile Ultrapure water (CLRW)
according to the following table:

Note: Use the appropriate buffer supplied by the enzyme manufacturer. For NEB enzymes, the corresponding buffer for
AscI and ApaI is NEBuffer 4. For Roche the corresponding buffer for ApaI is buffer A. Roche does not sell AscI.

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory

180 μl 1800 μl 2700 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl

4
Directions for making and testing PFGE plugs of Salmonella ser. Braenderup H9812 are in PNL05.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 5 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

Total Volume 200 μl 2000 μl 3000 μl

b. Add 200 μl diluted restriction buffer (1X) to labeled 1.5 ml microcentrifuge tubes.
c. Carefully remove plug from TE with spatula and place in a sterile disposable Petri dish or on large glass slide.
d. Cut a 2.0 to 2.5 mm-wide slice from each test sample and the appropriate number of S. ser. Braenderup H9812
standards with a single edge razor blade (or scalpel, cover slip, etc.) and transfer to tube containing diluted
restriction buffer. Be sure plug slice is under buffer. Replace rest of plug in the original tube that contains 5
ml TE buffer and store at 4ºC.

Note: PulseNet recommends that the combs with larger teeth (10 mm-wide teeth) be used to cast the gels because
computer analysis of the gel lanes is more accurate and less tedious than analysis of gel lanes cast with combs with the
smaller teeth (5.5 mm). Using combs with smaller teeth is not advised. The number of slices that can be cut from the
plugs will depend on the skill and experience of the operator, integrity of the plug, and whether the slices are cut
vertically or horizontally (plugs made in disposable molds).

e. Incubate sample and control plug slices in water bath or incubator for 5-10 min or at room temperature for 10-
15 min.
i. Incubate samples to be restricted with AscI and XbaI at 37°C.
ii. Incubate samples to be restricted with ApaI at 30°C (Roche) or 25°C (New England Biolabs).
f. After incubation, remove buffer from plug slice using a pipet fitted with 200-250 μl tip all the way to bottom of
tube and aspirate buffer. Be careful not to damage the plug slice with pipet tip and that plug slice is not
discarded with pipet tip.

2. Prepare the restriction enzyme master mix according to the following table. May mix in the same tube that was
used for the diluted restriction buffer:

Note: Enzymes may be purchased in several different stock concentrations. The calculations outlined here are based on
using an enzyme at a concentration of 10 or 50 U/μl. If the enzyme used is of a different concentration, make necessary
adjustments to the volume of enzyme and water to achieve a final concentration of 25 U/ sample.

Note: Keep vial of restriction enzyme on ice or in insulated storage box (-20˚C) at all times.

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory
175.5 μl 1755 μl 2632.5 μl
Reagent Water (CLRW)
10X Corresponding
20 μl 200 μl 300 μl
Restriction Buffer
BSA (10mg/ml) 2 μl 20 μl 30 μl
AscI (10 U/μl) 2.5 μl 25 μl 37.5 μl

Total Volume 200 μl 2000 μl 3000 μl

Reagent μl/Plug Slice µl/10 Plug Slices μl/15 Plug Slices

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 6 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

Sterile Clinical Laboratory

177.5 μl 1775 μl 2662.5 μl
Reagent Water (CLRW)
10X Corresponding
20 μl 200 μl 300 μl
Restriction Buffer
BSA (10mg/ml) 2 μl 20 μl 30 μl
ApaI (50 U/μl) 0.5 μl 5 μl 7.5 μl

Total Volume 200 μl 2000 μl 3000 μl

Note: Addition of Bovine Serum Albumin (BSA; highly recommended): Several restriction enzyme vendors
specifically recommend the addition of 1X BSA to enzyme restriction mixtures while others do not. PulseNet
Central recommends adding BSA to all enzyme restriction mixtures to minimize the incidence of incomplete
restriction.

3. Add 200 μl restriction enzyme master mix to each tube. Close tube and mix by tapping gently; be sure plug slices
are under enzyme mixture.

4. Incubate sample and standard (control) plug slices for 2 hours in a water bath at the appropriate temperature for
the enzyme.
a. Incubate samples restricted with ApaI at 25°C.
b. Incubate samples restricted with AscI and XbaI at 37°C.

5. If plug slices will be loaded into the wells (Option B, page 9), continue with Steps 1-4 of the next section
(CASTING AGAROSE GEL) approximately 1 h before restriction digest reaction is finished so the gel can
solidify for at least 30 minutes before loading the restricted PFGE plugs.

CASTING AGAROSE GEL

A. Loading Restricted Plug Slices on the Comb:

1. Confirm that water bath is equilibrated to 55-60ºC.

2. Make volume of 0.5X Tris-Borate EDTA Buffer (TBE) that is needed for both the gel and electrophoresis running
buffer according to one of the following tables.

5X TBE:
Reagent Volume in milliliters (ml)
5X TBE 200 220
Clinical Laboratory Reagent
1800 1980
Water (CLRW)
Total Volume of 0.5X TBE 2000 2200

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 7 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

10X TBE:
Reagent Volume in milliliters (ml)
10X TBE 100 110
Clinical Laboratory Reagent
1900 2090
Water (CLRW)
Total Volume of 0.5X TBE 2000 2200

3. Make 1% SeaKem Gold (SKG) agarose in 0.5X TBE as follows:

a. Weigh appropriate amount of SKG into 500 ml screw-cap flask.
b. Add appropriate amount of 0.5X TBE; swirl gently to disperse agarose.
i. Mix 1.0 g agarose with 100 ml 0.5X TBE for 14 cm wide gel form (10 wells)
ii. Mix 1.5 g agarose with 150 ml 0.5X TBE for 21 cm wide gel form (15 wells)
c. Loosen cap and microwave for 60 sec; mix gently and repeat for 15 sec intervals until agarose is completely
dissolved.
d. Recap flask and return to 55-60ºC water bath and equilibrate the agarose in the water bath for 15 minutes or
until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: Agarose LFTM (Amresco, X174) is the only acceptable alternative to SeaKem Gold, at this time. The time and
temperature needed to completely dissolve the agarose is dependent on the specifications of the microwave used and will
have to be determined empirically in each laboratory. Similarly, the optimal running time for each agarose will have to
be determined empirically in each laboratory.

4. A small volume (2-5 ml) of melted and cooled (50-60ºC) 1% SKG agarose may be wanted to seal wells after plugs
are loaded. Prepare as described above. Unused SKG agarose can be kept at room temperature, melted, and reused
several times.

Note: Place the gel form on a leveling table and adjust until perfectly leveled. Place the comb holder so the front part
(side with small metal screws) and teeth face the bottom of gel frame and the comb teeth touch the gel platform.

5. Remove restricted plug slices from the water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE.
Incubate at room temperature for 5 min.

6. Remove plug slices from tubes; put comb on bench top and load plug slices on the bottom of the comb teeth as
follows:
a. Load S. ser. Braenderup H9812 standards on teeth (lanes) 1, 5, 10 (10-well gel) or on teeth 1, 5, 10, 15 (15-well
gel).
b. Load samples on remaining teeth and note locations.

7. Remove excess buffer with tissue or kimwipe. Allow plug slices to air dry on the comb for 5-10 minutesor seal
them to the comb with 1% SKG agarose (55-60ºC).

8. Position comb in leveled gel form and confirm that the plugs slices are correctly aligned on the bottom of the comb
teeth, and that the lower edge of the plug slice is flush against the black platform..

9. Carefully pour the agarose (cooled to 55-60ºC) into the gel form and remove any bubbles or debris.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 8 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

10. Put black gel frame in electrophoresis chamber. Add 2 -2.2 L freshly prepared 0.5X TBE. Close cover of unit. The
amount of buffer needed depends on whether residual buffer was left in tubing or if unit was flushed with water
after the last gel was run.

11. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute (setting of ~70) and cooling module (14ºC)
approximately 30 minutes before gel is to be run.

12. Remove comb after gel solidifies, about 30-45 minutes.

13. Fill in wells of gel with melted and cooled (55-60ºC) 1% SKG Agarose (optional). Unscrew and remove end gates
from gel form; remove excess agarose from sides and bottom of casting platform with a tissue or kimwipe. Keep
gel on casting platform and carefully place gel inside black gel frame in electrophoresis chamber. Close cover of
chamber.

B. Loading Restricted Plug Slices into the Wells:

1. Follow steps 1-4 in Option A on pages 7 and 8 (Loading Restricted Plug Slices on the Comb).

Note: Place the gel form on a leveling table and adjust until perfectly leveled before pouring gel. Position the comb
holder so that front part (side with small metal screws) and teeth face the bottom of gel and the bottom edge of the
comb is 2 mm above the surface of the gel platform.

2. Cool melted SKG agarose in 55-60ºC water bath for 15-20 min; carefully pour agarose into gel form (casting stand)
fitted with comb. Be sure there are no bubbles.

3. Put black gel frame in electrophoresis chamber. Add 2-2.2 L freshly prepared 0.5X TBE. Close cover of unit. (The
amount of buffer depends on whether residual buffer was left in tubing, or if unit was flushed with water after the
last gel was run.)

4. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute (set at ~70) and cooling module (14ºC)
approximately 30 min before gel is to be run.

5. Remove restricted plug slices from 37ºC water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE.
Incubate at room temperature for 5 minutes.

6. Remove comb after gel solidifies for at least 30 minutes.

7. Remove restricted plug slices from tubes with tapered end of spatula and load into appropriate wells. Gently push
plugs to bottom and front of wells with wide end of spatula. Manipulate position with spatula and be sure that are
no bubbles.
a. Load S. ser. Braenderup H9812 standards in wells (lanes) 1, 5, 10 (10 well gel) or in wells 1, 5, 10, 15 (15 well
gel).
b. Load samples in remaining wells.

Note: Loading the plug slices can be tedious; each person has to develop his/her own technique for consistently placing
the plug slices in the wells so the lanes will be straight and the bands sharp.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 9 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

8. Fill in wells of gel with melted 1% SKG agarose (equilibrated to 55-60ºC). Allow to harden for 3-5 min. Unscrew
and remove end gates from gel form; remove excess agarose from sides and bottom of casting platform with a tissue
or kimwipe. Keep gel on casting platform and carefully place gel inside black gel frame in electrophoresis chamber.
Close cover of chamber.

ELECTROPHORESIS CONDITIONS

1. Select following conditions for Listeria monocytogenes strains restricted with AscI or ApaI:
a. Select following conditions on CHEF Mapper
Auto Algorithm
49 kb - low MW
450 kb - high MW
Select default values except where noted by pressing "enter."
Initial switch time = 4.0 s
Final switch time = 40.0 s
Change run time to 18 - 19 h (See note below)

b. Select following conditions on CHEF-DR III

Initial switch time: 4.0 s
Final switch time: 40.0 s
Voltage: 6 V
Included Angle: 120°
Run time: 18-19 h (See note below)

c. Select following conditions on CHEF-DR II

Initial A time: 4.0 s
Final A time: 40.0 s
Start ratio: 1.0 (if applicable)
Voltage: 200 V
Run time: 19-20 h (See note below)

Note: The electrophoresis running times recommended above are based on the equipment and reagents used at the CDC.
Run times may be different in your laboratory and will have to be optimized for your gels so that the lowest band
in the S. ser. Braenderup H9812 standard migrates 1.0 - 1.5 cm from the bottom of the gel.

Note: Make note of the initial milliamp (mAmp) reading on the instrument. The initial mAmps should be between 110-
150 mAmps. A reading outside of this range may indicate that the 0.5X TBE buffer was prepared improperly and the
buffer should be remade.

Day 2

STAINING AND DOCUMENTATION OF PFGE AGAROSE GEL

Note: The following staining procedure describes the use of ethidium bromide to stain PFGE gels. Alternate DNA
stains may be used. Please see the “Alternate DNA Stains-Results and Recommendations” posting within the PulseNet
Documents forum on the SharePoint site for additional information.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 10 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

1. When electrophoresis run is over, turn off equipment; remove and stain gel with ethidium bromide by diluting 40 μl
of ethidium bromide stock solution (10 mg/ml) with 400 ml CLRW. This volume is for a staining box that is
approximately 14 cm x 24 cm; a larger container may require a larger amount of staining solution). Stain gel for 20
- 30 min in covered container.

Note: Ethidium bromide is toxic and a mutagen. Stock solutions of 10 mg/ml Ethidium Bromide (EtBr) in water are
available from several commercial companies (Amresco X328; Bio-Rad, 161-0433; Sigma, E-1510). The diluted
solution can be kept in dark bottle and reused 6-8 times before discarding according to your institution's guidelines for
hazardous waste. CDC does not recommend disposing of EtBr down the drain. Aqueous solutions containing EtBr
can be filtered through charcoal or degraded using activated carbon destaining or “tea” bags from Amresco (E732-
25 Destaining Bags) or other companies, which effectively and safely remove EtBr from solutions and gels. Once
the EtBr is removed, the treated aqueous solutions can be discarded down the drain. If you have further questions
about EtBr please refer to the Material Safety Data Sheets (MSDS) provided by the vendor or manufacturer.

Note: Currently, the only acceptable alternative stain options are GelRedTM (Biotium, 31010), SYBR® Safe
(Invitrogen, S-33102) and SYBR® Gold (Invitrogen, S-11494). Labs are strongly encouraged to follow
manufacturer’s instructions and test stains in their labs before adopting them for routine use. If one of the
alternative stains is used, the destaining steps should be omitted.

2. Destain gel in approximately 500 ml CLRW for 60 - 90 min, changing water every 20 minutes. Capture image using
a Gel Doc 1000, 2000, EQ, XR, or equivalent documentation system. If too much background is observed destain
for an additional 30-60 min.

3. Follow directions given with the imaging equipment to save gel image as a *.1sc file; convert this file to *.tif file for
analysis with the BioNumerics software program. The gel image should fill the entire window of the imaging
equipment (computer) screen (without cutting off wells or lower bands). Ensure that the image is in focus and
that there is little to no saturation (over-exposure) in the bands (signified by red pixilation in the QuantityOne or
ImageLab software). Additional instructions are provided in PNL07 of the PulseNet QA/QC manual.

4. Drain buffer from electrophoresis chamber and discard. Rinse chamber with 2 L CLRW or, if unit is not going to be
used for several days, flush lines with water by letting pump run for 5-10 min before draining water from chamber
and hoses.

5. If the lowest band in the H9812 standard does not migrate within 1-1.5 cm of the bottom of the gel, the proper run
time will need to be determined empirically for the conditions in each laboratory.

Note: The following options are available if PFGE results do not have to be available within 24-28 hours:
− Plugs can be lysed for longer periods of time (3-16 hours).
− The washing steps with TE to remove the lysis buffer from the PFGE plugs can be done for longer periods
of time (30-45 min) and at lower temperatures (37°C or room temperature). They can be started on Day 1
and finished on Day 2 after overnight refrigeration of the plugs in TE.

Use of trade names and commercial sources is for identification purposes only and does not imply endorsement by CDC
or the U.S. Department of Health and Human Services.

NOTE: CLIA LABORATORY PROCEDURE MANUAL REQUIREMENTS

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 11 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

Efforts have been made to assure that the procedures described in this protocol have been written in accordance with
the 1988 Clinical Laboratory Improvement Amendments (CLIA) requirements for a procedure manual (42 CFR
493.1211). However, due to the format required for training, the procedures will require some modifications and
additions to customize them for your particular laboratory operation.
Any questions regarding the CLIA requirements for a procedure manual, quality control, quality assurance, etc.,
should be directed to the agency or accreditation organization responsible for performing your laboratory's CLIA
inspection. In addition, some states and accreditation organizations may have more stringent requirements that will need
to be addressed.

Formulas of Selected Reagents used in PulseNet Standardized Laboratory Protocol for PFGE

Tris:EDTA Buffer, pH 8.0 (TE, 10 mM Tris:1 mM EDTA, pH 8.0)

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure Water (CLRW)

Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosine + 0.1 mg/ml Proteinase K)
25 ml (50 ml) of 1 M Tris, pH 8.0
50 ml (100 ml) of 0.5 M EDTA, pH 8.0
50 ml (100 ml) 10% N-Lauroylsarcosine, Sodium salt (Sarcosyl)
or
5 g (10 g) of N-Lauroylsarcosine, Sodium salt (Sarcosyl) 5
Dilute to 500 ml (1000 ml) with Sterile Ultrapure Water (CLRW)

Add 25 μl Proteinase K stock solution (20 mg/ml) per 5 ml of cell lysis buffer just before use for a final
concentration in the lysis buffer of 0.1 mg/ml Proteinase K.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:
8.1 The above protocol was revised and distributed on October 30, 2008, modifications as follows:
− Reduction of cell suspension concentration
− Solubilization of the Lysozyme in TE rather than water
− Volumes used for plug preparation
− Incubation of the cell suspensions with the Lysozmye for 10-20 minutes in a 56ºC water bath rather
than 37ºC
− Restriction enzyme units used for ApaI (50 U/sample) and AscI (40 U/ sample) for two to three hours
8.2 2011-07 A note was added to clarify which buffers to use during the restriction digestion “step 2” under the
enzyme mixture charts.

5
If Sarcosyl powder is added directly to the other components of this reagent, warm the solution to 50-60ºC
for 30-60 minutes, or leave at room temperature for ≈2 hours to completely dissolve the Sarcosyl; adjust to the
final volume with sterile Sterile Clinical Laboratory Reagent Water.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 12 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE CODE: PNL04
Effective Date:
OF LISTERIA MONOCYTOGENES 03 24 2013

8.3 2013-03 changes:

− Corrected formula for TE buffer. TE used at CDC is 10mM for Tris and 1mM for EDTA.
− Recommended disinfectant changed from 10% bleach to 1% Lysol/Amphyll or 90% ethanol.
− Corrected calculations for plug agarose.
− New options for Lysozyme were added because Sigma L7651 was discontinued.
− A note was added to provide guidance when working with large numbers of isolates (>10).
− Volume of TE needed for washing plugs was corrected from 300 – 350 ml to 400 – 600 ml.
− Use of pre-restriction step was changed from optional to highly recommended.
− A statement was added to clarify that using combs with smaller teeth (5.5 mm) was not advised.
− Units of enzyme for AscI and ApaI was reduced from 40 units and 50 units, respectively, to 25 units
for each. Incubation time was changed to 2 hours, instead of a range of 2 – 3 hours.
− A statement was included to allow the use of an alternative agarose for casting the running gel, along
with recommendations strongly urging each lab to optimize the run time. Internal and external
validation showed that run times could be affected by agarose type, but no trends were noted so a
blanket recommendation on run times cannot be made. Additional agarose alternatives may be tested
and deemed acceptable at a later date.
− A statement was included to allow the use of alternative DNA stains that are equivalent to EtBr. Labs
are strongly urged to follow manufacturer’s instructions as well as test stains in their own labs to gain
experience using alternative agarose stains. Additional stain alternatives may be tested and deemed
acceptable at a later date.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 13 of 13
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

1. PURPOSE: To describe the One-Day (24-26 h) Standardized Laboratory Protocol for Molecular
Subtyping of E. coli O157:H7, E. coli Non-O157 (STEC), Salmonella, Shigella sonnei and Shigella
flexneri by Pulsed-field Gel Electrophoresis (PFGE).

2. SCOPE: To provide the PulseNet participants with a standardized procedure for performing PFGE
of E. coli O157:H7, E. coli Non-O157 (STEC), Salmonella, Shigella sonnei and Shigella flexneri,
thus ensuring inter-laboratory comparability of the generated results.

3. DEFINITIONS/TERMS:
3.1 PFGE: Pulsed-field Gel Electrophoresis
3.2 DNA: Deoxyribonucleic acid
3.3 CDC: Centers for Disease Control and Prevention
3.4 CLRW: Clinical Laboratory Reagent Water

4. RESPONSIBILITIES/PROCEDURE:

BIOSAFETY WARNING: Escherichia coli O157:H7, Salmonella serotypes, Shigella sonnei, and Shigella flexneri are
human pathogens and can cause serious disease. It has been reported that less than 100 cells of E. coli O157:H7 may
cause infection. Shigella species also have a low infectious dose and are demonstrated hazards to laboratory personnel.
Always use Biosafety Level 2 practices (at a minimum) and extreme caution when transferring and handling strains of
these genera. Work in a biological safety cabinet when handling large amounts of cells. Disinfect or dispose of all
plasticware and glassware that come in contact with the cultures in a safe manner.

Please read all instructions carefully before starting protocol. Treat all plasticware, glassware, pipets, spatulas, etc. that
come in contact with the cell suspensions or plugs as contaminated materials and dispose of, or disinfect according to the
guidelines of your institution. Disinfect reusable plug molds before they are washed; the disposable plug molds,
including the tape and the tab that is used to push the plugs out of the wells, are also contaminated and should be
disinfected 1% Lysol/Amphyll or 90% ethanol for at least 30 minutes if they will be washed and reused.

Day 0
Streak an isolated colony from test cultures onto Trypticase Soy Agar with 5% defibrinated sheep blood (TSA-SB)
plates (or comparable non-selective media) for confluent growth. It is recommended that a storage vial of each culture be
created. To do this stab small screw cap tubes of TSA, HIA, or similar medium with the same inoculating loop used to
streak the plate. This will ensure that the same colony can be retested if necessary. Incubate cultures at 37ºC for 14-18 h.

Day 1
1. Turn on shaker water bath or incubator (54-55ºC), stationary water baths (55-60ºC) and spectrophotometer
(or equivalent instrument such as the Dade Microscan Turbidity meter or bioMérieux Vitek colorimeter).

2. Prepare TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0)1 as follows:

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure Clinical Laboratory Reagent Water (CLRW)

Note: The TE Buffer is used to make the plug agarose and also to wash lysed PFGE plugs.

1
Additional information is found on page 13 of this document.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 1 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

3. Prepare 1% SeaKem Gold agarose in TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) for PFGE plugs as follows:
a. Weigh 0.50 g (or 0.25 g) SeaKem Gold (SKG) agarose into 250 ml screw-cap flask.
b. Add 50.0 ml (or 25.0 ml) TE Buffer; swirl gently to disperse agarose.
c. Loosen or remove cap, cover loosely with clear film, and microwave for 30sec; mix gently and repeat for 10sec
intervals until agarose is completely dissolved.
d. Recap flask and return to 55-60ºC water bath and equilibrate the agarose in the water bath for 15 minutes or
until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: SeaKem Gold agarose works well for making PFGE plugs because it provides added strength to the plugs
minimizing breakage of plugs during the lysis and washing steps. The time and temperature needed to completely
dissolve the agarose is dependent on the specifications of the microwave used, and will have to be determined
empirically in each laboratory.

4. Label small transparent tubes (12 mm x 75 mm Falcon 2054 tubes or equivalent) with culture numbers.

5. Prepare Cell Suspension Buffer (100 mM Tris:100 mM EDTA, pH 8.0) as follows:

10 ml of 1 M Tris, pH 8.0
20 ml of 0.5 M EDTA, pH 8.0
Dilute to 100 ml with sterile Ultrapure water (CLRW)

6. Transfer ≈2 ml of Cell Suspension Buffer (CSB) to small labeled tubes. Use a sterile polyester-fiber or cotton
swab that has been moistened with sterile CSB to remove some of the growth from agar plate; suspend cells in CSB
by spinning swab gently so cells will be evenly dispersed and formation of aerosols is minimized.

Note: The minimum volume of the cell suspension needed will depend on size of the cuvettes or tubes used to measure
the cell concentration and are dependent on the manufacturer’s specifications for the spectrophotometer, turbidity meter,
or colorimeter. Keep suspensions on ice if you have more than 6 cultures to process or refrigerate cell suspensions if you
cannot adjust their concentration immediately.

7. Adjust concentration of cell suspensions to one of values given below by diluting with sterile CSB or by adding
additional cells.
a. Spectrophotometer: 610 nm wavelength, absorbance (Optical Density) of 1.00 (range of 0.8-1.0)
b. Dade Microscan Turbidity Meter: 0.40 - 0.45 (measured in Falcon 2054 tubes)
0.58 - 0.63 (measured in Falcon 2057 tubes)
c. bioMérieux Vitek colorimeter: ≈17-18% transmittance (measured in Falcon 2054 tubes)

Note: The values in Steps 7a, 7b and 7c give satisfactory results at CDC; each laboratory may need to establish the
optimal concentration needed for satisfactory results.

CASTING PLUGS

Label wells of PFGE plug molds with culture number. When reusable plug molds are used, put strip of tape on lower
part of reusable plug mold before labeling wells.

Note: Unused plug agarose can be kept at room temperature and reused 1-2 times. Microwave on low-medium power
for 10 -15 sec and mix; repeat for 5-10 sec intervals until agarose is completely melted. This agarose melts rapidly!

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 2 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

Note: Proteinase K solutions (20 mg/ml) are available commercially. Alternatively, a stock solution of Proteinase K can
be prepared from the powder in sterile Ultrapure water (CLRW). For best results, aliquot in 300-500 μl into small tubes
and store in a freezer (-20ºC) until ready to use. Just before use, thaw appropriate number of vials needed for the
samples; keep Proteinase K solutions on ice. If the Proteinase K stock solution was prepared from powder, discard any
thawed solution at the end of the work day. Store commercially prepared Proteinase K solutions according to directions
provided by the supplier.

1. Transfer 400 μl (0.4 ml) adjusted cell suspensions to labeled 1.5 ml microcentrifuge tubes.

2. Add 20 μl of Proteinase K (20 mg/ml stock) to each tube and mix gently with pipet tip. (200 μl are needed for 10
cell suspensions.)

3. Add 400 μl melted 1% SeaKem Gold agarose to 400 μl cell suspension; mix by gently pipetting mixture up and
down a few times. Over-pipeting can cause DNA shearing. Maintain temperature of melted agarose by keeping
flask in beaker of warm water (55-60ºC).

4. Immediately, dispense part of mixture into appropriate well(s) of reusable plug mold. Do not allow bubbles to form.
Two plugs of each sample can be made from these amounts of cell suspension and agarose and are useful if repeat
testing is required. Allow plugs to solidify at room temperature for 10-15 min. They can also be placed in the
refrigerator (4ºC) for 5 minutes

Note: If disposable plug molds are used for making plugs with 1% SeaKem Gold agarose, use 200 μl cell suspension,
10 μl of Proteinase K (20 mg/ml stock) and 200 μl of agarose; up to 4 plugs can be made from these amounts of cell
suspension and agarose.

Note: The generation of cell suspension and the subsequent casting of the plugs should be performed as rapidly as
possible in order to minimize premature cell lysis. If large numbers of samples are being prepared, it is recommended
that they be processed in batches of ~10 samples at a time. Once the first batch of isolates are in the cell lysis incubation,
then start preparing the cells suspensions the next group samples, and so on. All batches can be lysed and washed
together, since additional lysis time will not affect the initial batches.

LYSIS OF CELLS IN AGAROSE PLUGS

Note: Two plugs (reusable molds) or up to four plugs (disposable molds) of the same strain can be lysed in the same
50ml tube.

1. Label 50ml polypropylene screw-cap or 50ml Oak Ridge tubes with culture numbers.

2. Prepare Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) as follows:
25 ml of 1 M Tris, pH 8.0
50 ml of 0.5 M EDTA, pH 8.0
50 ml of 10 % Sarcosyl (N-Lauroylsarcosine, Sodium salt) 2
Dilute to 500 ml with sterile Ultrapure water (CLRW)

3. Calculate the total volume of Cell Lysis/Proteinase K Buffer needed as follows:

2
The N-Lauroylsarcosine, Sodium salt can be added directly to the other ingredients and allowed to dissolve. See
page 13 of this document.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 3 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

a. 5 ml Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) is needed per tube
(e. g., 5 ml x 10 tubes = 50 ml).
b. 25 μl Proteinase K stock solution (20 mg/ml) is needed per tube of the cell lysis buffer
(e. g., 25 μl x 10 tubes = 250 μl).
c. Prepare the master mix by measuring the correct volume of Cell Lysis Buffer and Proteinase K into appropriate
size test tube or flask and mix well.

Note: The final concentration of Proteinase K in the lysis buffer is 0.1 mg/ml and is different from the concentration that
was added to the cell suspension (0.5 mg/ml).

4. Add 5 ml of Proteinase K/Cell Lysis Buffer to each labeled 50 ml tube.

5. Trim excess agarose from top of plugs with scalpel, razor blade or similar instrument. Open reusable plug mold and
transfer plugs from mold with a 6-mm wide spatula to appropriately labeled tube. If disposable plug molds are used,
remove white tape from bottom of mold and push out plug(s) into appropriately labeled tube. Be sure plugs are
under buffer and not on side of tube.

Note: The excess agarose, plug mold, spatula, etc. are contaminated. Discard or disinfect appropriately.

6. Remove tape from reusable mold. Place both sections of the plug mold, spatulas, and scalpel in 90% ethanol, 1%
Lysol/Amphyll other suitable disinfectant. Soak them for 15 minutes before washing them. Discard disposable
plug molds or disinfect them in or 90% ethanol for 30-60 minutes if they will be washed and reused.

7. Place tubes in rack and incubate in a 54-55ºC shaker water bath or incubator for 1.5 - 2 h with constant and
vigorous agitation (150-175 rpm). If lysing in water bath, be sure water level is above level of lysis buffer in tubes.

8. Pre-heat enough sterile Ultrapure water (CLRW) to 54-55ºC so that plugs can be washed two times with 10-15 ml
water (200-250 ml for 10 tubes).

WASHING OF AGAROSE PLUGS AFTER CELL LYSIS

Note: Most laboratories will find that their plugs are sufficiently stable to perform the following washing steps at
54-55ºC. However, if you notice that your plugs are nicked along the edges or breaking it will be necessary for your
laboratory to lower the water bath or incubator to 50ºC for the following washing steps.

1. Remove tubes from water bath or incubator, and carefully pour off lysis buffer into an appropriate discard container;
plugs can be held in tubes with a screened cap or spatula.

Note: It is important to remove all of the liquid during this and subsequent wash steps by touching edge of tube or
screened cap on an absorbent paper towel.

2. Add at 10-15 ml sterile Ultrapure water (CLRW) that has been pre-heated to 54-55ºC to each tube and shake the
tubes in a 54-55ºC water bath or incubator for 10-15 min.

3. Pour off water from the plugs and repeat wash step with pre-heated water (Step 2) one more time.
a. Pre-heat enough sterile TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) in a 54-55ºC water bath so that plugs
can be washed four times with 10-15 ml TE (400-600 ml for 10 tubes) after beginning last water wash.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 4 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

4. Pour off water, add 10-15 ml pre-heated (54-55ºC) sterile TE Buffer, and shake the tubes in 54-55ºC water bath or
incubator for 10-15 min.

5. Pour off TE and repeat wash step with pre-heated TE three more times.

6. Decant last wash and add 5-10 ml sterile TE. Continue with step 1 in "Restriction Digestion" section or store plugs
in TE Buffer at 4ºC until needed. Plugs can be transferred to smaller tubes for long term storage.

Note: If restriction digestion is to be done the same day, complete Steps 1-3 of next section (Restriction Digestion)
during last TE wash step for optimal use of time.

RESTRICTION DIGESTION OF DNA IN AGAROSE PLUGS

Note: A small slice of the plug (not the entire plug) should be digested with the primary restriction enzyme because less
enzyme is required and other slices of the plug can be subjected to restriction analysis with secondary or tertiary
enzymes, according to the table below. E. coli species, Salmonella, and Shigella sonnei utilize XbaI as the primary
enzyme and BlnI as the secondary enzyme. Shigella flexneri are tested with NotI as the primary enzyme and XbaI as the
secondary enzyme. The use of a secondary (or tertiary) enzyme is useful in situations where the PFGE patterns obtained
with the primary enzyme from two or more isolates are indistinguishable.

Primary Enzyme Secondary Enzyme Tertiary Enzyme

Organism
(Concentration) (Concentration) (Concentration
XbaI BlnI/AvrII SpeI
E. coli O157
(50 U/sample) (30U/sample) (30 U/sample)
XbaI BlnI/AvrII SpeI
E. coli non-O157
(50 U/sample) (30U/sample) (30 U/sample)
XbaI BlnI/AvrII SpeI
Salmonella
(50 U/sample) (30U/sample) (30 U/sample)
XbaI BlnI/AvrII SpeI
Shigella sonnei
(50 U/sample) (30U/sample) (30 U/sample)
NotI XbaI SpeI
Shigella flexneri
(50 U/sample) (50 U/ sample) (30 U/sample)

1. Label 1.5 ml microcentrifuge tubes with culture numbers; label 3 (10-well gel) or 4 (15-well gel) tubes for
Salmonella ser. Braenderup H9812 standards.
a. Pre-Restriction Incubation Step (highly recommended): Prepare a master mix by diluting the appropriate
10X restriction buffer (Roche Applied Science or equivalent) 1:10 with sterile Ultrapure water (CLRW)
according to the following table:

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory

180 μl 1800 μl 2700 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl
Total Volume 200 μl 2000 μl 3000 μl

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 5 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

b. Add 200 μl diluted restriction buffer (1X) to labeled 1.5 ml microcentrifuge tubes.
c. Carefully remove plug from TE with spatula and place in a sterile disposable Petri dish or on large glass slide.
d. Cut a 2.0 to 2.5 mm wide slice from each test samples and the appropriate number of S. ser. Braenderup H9812
standards with a single edge razor blade (or scalpel, cover slip, etc.) and transfer to tube containing diluted
restriction buffer. Be sure plug slice is under buffer. Replace rest of plug into the original tube that contains
5 ml TE buffer and store at 4ºC.

Note: PulseNet recommends that the combs with larger teeth (10 mm wide teeth) be used to cast the gels because
computer analysis of the gel lanes is more accurate and less tedious than analysis of gel lanes cast with combs with the
smaller teeth (5.5 mm). Using combs with smaller teeth is not advised. The number of slices that can be cut from the
plugs will depend on the skill and experience of the operator, integrity of the plug, and whether the slices are cut
vertically or horizontally (plugs made in disposable molds).

e. Incubate sample and control plug slices in a 37ºC water bath for 5-10 min or at room temp for 10-15 min.
f. After incubation, remove buffer from plug slice using a pipet fitted with 200-250 μl tip all the way to bottom of
tube and aspirate buffer. Be careful not to damage the plug slice with pipet tip and that plug slice is not
discarded with pipet tip.

2. Prepare the restriction enzyme master mix according to the following table3. May mix in the same tube that was
used for the diluted restriction buffer.

Note: Enzymes may be purchased in several different stock concentrations. The calculations outlined here are based on
using an enzyme at a concentration of 10 U/μl. If the enzyme used is of a different concentration, make necessary
adjustments to the volume of enzyme and water to achieve a final concentration of 50 U/ sample.

Note: Keep vial of restriction enzyme on ice or in insulated storage box (-20˚C) at all times.

Reagent μl/Plug Slice µl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory
173 μl 1730 μl 2595 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl
BSA (10mg/ml) 2 μl 20 μl 30 μl
Enzyme (10 U/μl) 5 μl 50 μl 75 μl

Total Volume 200 μl 2000 μl 3000 μl

Note: Addition of Bovine Serum Albumin (BSA; highly recommended): Several restriction enzyme vendors
specifically recommend the addition of 1X BSA to enzyme restriction mixtures while others do not. PulseNet
Central recommends adding BSA to all enzyme restriction mixtures to minimize the incidence of incomplete
restriction.

3. Add 200 μl restriction enzyme master mix to each tube. Close tube and mix by tapping gently; be sure plug slices
are under enzyme mixture.

4. Incubate sample and control plug slices in 37˚C water bath for 1.5-2 h.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 6 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

5. If plug slices will be loaded into the wells (Option B, page 8), continue with Steps 1-4 of the next section
(CASTING AGAROSE GEL) approximately 1 h before restriction digest reaction is finished so the gel can
solidify for at least 30 minutes before loading the restricted PFGE plugs.

CASTING AGAROSE GEL

A. Loading Restricted Plug Slices on the Comb:

1. Confirm that water bath is equilibrated to 55-60ºC.

2. Make volume of 0.5X Tris-Borate EDTA Buffer (TBE) that is needed for both the gel and electrophoresis running
buffer according to one of the following tables.

5X TBE:
Reagent Volume in milliliters (ml)
5X TBE 200 220
Clinical Laboratory Reagent
1800 1980
Water (CLRW)
Total Volume of 0.5X TBE 2000 2200

10X TBE:
Reagent Volume in milliliters (ml)
10X TBE 100 110
Clinical Laboratory Reagent
1900 2090
Water (CLRW)
Total Volume of 0.5X TBE 2000 2200

3. Make 1% SeaKem Gold (SKG) Agarose in 0.5X TBE as follows:

a. Weigh appropriate amount of SKG into 500 ml screw-cap flask.
b. Add appropriate amount of 0.5X TBE; swirl gently to disperse agarose.
i. Mix 1.0 g agarose with 100 ml 0.5X TBE for 14 cm wide gel form (10 wells)
ii. Mix 1.5 g agarose with 150 ml 0.5X TBE for 21 cm wide gel form (15 wells)
c. Loosen or remove cap and cover loosely with clear film, and microwave for 60 sec; mix gently and repeat for
15 sec intervals until agarose is completely dissolved.
d. Recap flask and return to 55-60ºC water bath and equilibrate the agarose in the water bath for 15 minutes or
until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: Agarose LFTM (Amresco, X174) is the only acceptable alternative to SeaKem Gold, at this time. The time and
temperature needed to completely dissolve the agarose is dependent on the specifications of the microwave used and will
have to be determined empirically in each laboratory. Similarly, the optimal running time for each agarose will have to
be determined empirically in each laboratory.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 7 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

4. A small volume (2-5 ml) of melted and cooled (55-60ºC) 1% SKG agarose may be wanted to seal wells after plugs
are loaded. Prepare as described above. Unused SKG agarose can be kept at room temperature, melted, and reused
several times.

Note: Place the gel form on a leveling table and adjust until perfectly leveled. Place the comb holder so the front part
(side with small metal screws) and teeth face the bottom of gel frame and the comb teeth touch the gel platform.

5. Remove restricted plug slices from 37ºC water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE.
Incubate at room temperature for 5 min.

6. Remove plug slices from tubes; put comb on bench top and load plug slices on the bottom of the comb teeth as
follows:
a. Load S. ser. Braenderup H9812 standards on teeth (lanes) 1, 5, 10 (10 well gel) or on teeth 1, 5, 10, 15 (15 well
gel).
b. Load samples on remaining teeth and note locations.

7. Remove excess buffer with tissue or kimwipe. Allow plug slices to air dry on the comb for 5-10 minutes or seal
them to the comb with 1% SKG agarose (55-60ºC).

8. Position comb in leveled gel form and confirm that the plugs slices are correctly aligned on the bottom of the comb
teeth, and that the lower edge of the plug slice is flush against the black platform.

9. Carefully pour the agarose (cooled to 55-60ºC) into the gel form and remove any bubbles or debris.

10. Put black gel frame in electrophoresis chamber. Add 2-2.2 L freshly prepared 0.5X TBE. Close cover of unit. The
amount of buffer needed depends on whether residual buffer was left in tubing or if unit was flushed with water
after the last gel was run.

11. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute (setting of ≈70) and cooling module (14ºC)
approximately 30 minutes before gel is to be run.

12. Remove comb after gel solidifies, about 30-45 minutes.

13. Fill in wells of gel with melted and cooled (55- 60ºC) 1% SKG Agarose (optional). Unscrew and remove end gates
from gel form; remove excess agarose from sides and bottom of casting platform with a tissue or kimwipe. Keep
gel on casting platform and carefully place gel inside black gel frame in electrophoresis chamber. Close cover of
chamber.

B. Loading Restricted Plug Slices into the Wells:

1. Follow steps 1-4 in Section A on page 7-8 (Loading Restricted Plug Slices on the Comb).

Note: Place the gel form on a leveling table and adjust until perfectly leveled before pouring gel. Position the comb
holder so that front part (side with small metal screws) and teeth face the bottom of gel and the bottom edge of the
comb is 2 mm above the surface of the gel platform.

2. Cool melted SKG agarose in 55-60ºC water bath for 15-20 min; carefully pour agarose into gel form (casting stand)
fitted with comb. Be sure there are no bubbles.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 8 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

3. Put black gel frame in electrophoresis chamber. Add 2-2.2 L freshly prepared 0.5X TBE. Close cover of unit. (The
amount of buffer depends on whether residual buffer was left in tubing, or if unit was flushed with water after the
last gel was run).

4. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute (setting at ~70), and cooling module (14ºC)
approximately 30 minutes before gel is to be run.

5. Remove restricted plug slices from 37ºC water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE.
Incubate at room temperature for 5 minutes.

6. Remove comb after gel solidifies, about30 – 45 minutes.

7. Remove restricted plug slices from tubes with tapered end of spatula and load into appropriate wells. Gently push
plugs to bottom and front of wells with wide end of spatula. Manipulate position with spatula and be sure that are
no bubbles.
a. Load S. ser. Braenderup H9812 standards in wells (lanes) 1, 5, 10 (10 well gel) or in wells 1, 5, 10, 15 (15 well
gel).
b. Load samples in remaining wells.

Note: Loading the plug slices can be tedious; each person has to develop his/her own technique for consistently
placing the plug slices in the wells so the lanes will be straight and the bands sharp.

8. Fill in wells of gel with melted 1% SKG Agarose (equilibrated to 55-60ºC). Allow to harden for 3-5 min. Unscrew
and remove end gates from gel form; remove excess agarose from sides and bottom of casting platform with a tissue
or kimwipe. Keep gel on casting platform and carefully place gel inside black gel frame in electrophoresis chamber.
Close cover of chamber.

ELECTROPHORESIS CONDITIONS

1. Select following conditions for Escherichia coli O157:H7 and Shigella sonnei strains restricted with XbaI or AvrII
(BlnI):
a. Select following conditions on CHEF Mapper
Auto Algorithm
30 kb - low MW
600 kb - high MW
Select default values except where noted by pressing "enter."
Change run time to 18 - 19 h (See note below)
(Default values: Initial switch time = 2.16 s; Final switch time = 54.17 s)

b. Select following conditions on CHEF-DR III

Initial switch time: 2.2 s
Final switch time: 54.2 s
Voltage: 6 V
Included Angle: 120°
Run time: 18-19 h (See note below)

c. Select following conditions on CHEF-DR II

Initial A time: 2.2 s
Final A time: 54.2 s

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 9 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

Start ratio: 1.0 (if applicable)

Voltage: 200 V
Run time: 19-20 h (See note below)

2. Select following conditions for Salmonella strains restricted with XbaI or AvrII (BlnI):
a. Select following conditions on CHEF Mapper
Auto Algorithm
30 kb - low MW
700 kb - high MW
Select default values except where noted by pressing "Enter."
Change run time to 18 - 19 h (See note below)
(Default values: Initial switch time = 2.16 s; Final switch time = 63.8 s)

b. Select following conditions on CHEF DR-III

Initial switch time: 2.2 s
Final switch time: 63.8 s
Voltage: 6 V
Included Angle: 120°
Run time: 18-19 h (See note below)

c. Select following conditions on CHEF DR-II

Initial A time: 2.2s
Final A time: 63.8 s
Start Ratio: 1.0 (if applicable)
Voltage: 200 V
Run time: 19-20 h (See note below)

3. Select following conditions for Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) strains restricted with
XbaI or AvrII (BlnI):
a. Select following conditions on CHEF Mapper
Auto Algorithm
50 kb - low MW
400 kb - high MW
Select default values except where noted by pressing "Enter."
Change run time to 18 - 19 h (See note below)
(Default values: Initial switch time = 6.76 s; Final switch time = 35.38 s)

b. Select following conditions on CHEF DR-III

Initial switch time: 6.76 s
Final switch time: 35.38 s
Voltage: 6 V
Included Angle: 120°
Run time: 18-19 h (See note below)

c. Select following conditions on CHEF DR-II

Initial A time: 6.76 s
Final A time: 35.38 s
Start Ratio: 1.0 (if applicable)
Voltage: 200 V
Run time: 19-20 h (See note below)

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 10 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

4. Select following conditions for Shigella flexneri strains restricted with NotI or XbaI :
a. Select following conditions on CHEF Mapper
Auto Algorithm
50 kb - low MW
400 kb - high MW
Select default values except where noted by pressing "Enter."
Change the switch times to the following values:
Initial switch time: 5 seconds
Final switch time: 35 seconds
Change run time to 18 - 19 h (See note below)
(Default values: Initial switch time = 6.76 s; Final switch time = 35.38 s)

b. Select following conditions on CHEF DR-III

Initial switch time: 5 s
Final switch time: 35 s
Voltage: 6 V
Included Angle: 120°
Run time: 18-19 h (See note below)

c. Select following conditions on CHEF DR-II

Initial A time: 5 s
Final A time: 35 s
Start Ratio: 1.0 (if applicable)
Voltage: 200 V
Run time: 19-20 h (See note below)

Note: The electrophoresis running times recommended above are based on the equipment and reagents used at the CDC.
Run times may be different in your laboratory and will have to be optimized for your gels so that the lowest band
in the S. ser. Braenderup H9812 standard migrates 1.0 - 1.5 cm from the bottom of the gel.

Note: Make note of the initial milliamp (mAmp) reading on the instrument. The initial mAmps should be between 110-
150 mAmps. A reading outside of this range may indicate that the 0.5X TBE buffer was prepared improperly and the
buffer should be remade.

Day 2

STAINING AND DOCUMENTATION OF PFGE AGAROSE GEL

Note: The following staining procedure describes the use of ethidium bromide to stain PFGE gels. Alternate DNA
stains may be used. Please see the “Alternate DNA Stains-Results and Recommendations” posting within the PulseNet
Documents forum on the SharePoint site for additional information.

1. When electrophoresis run is over, turn off equipment; remove and stain gel with ethidium bromide by diluting 40 μl
of ethidium bromide stock solution (10 mg/ml) with 400 ml of Ultrapure water (CLRW). This volume is for a
staining box that is approximately 14 cm x 24 cm; a larger container may require a larger amount of staining
solution. Stain gel for 20-30 min in covered container.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 11 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

Note: Ethidium bromide is toxic and a mutagen. Stock solutions of 10 mg/ml Ethidium Bromide (EtBr) in water are
available from several commercial companies (Amresco X328; Bio-Rad, 161-0433; Sigma, E-1510). The diluted
solution can be kept in dark bottle and reused 6-8 times before discarding according to your institution's guidelines for
hazardous waste. CDC does not recommend disposing of EtBr down the drain. Aqueous solutions containing EtBr
can be filtered through charcoal or degraded using activated carbon destaining or “tea” bags from Amresco (E732-
25 Destaining Bags) or other companies, which effectively and safely remove EtBr from solutions and gels. Once
the EtBr is removed, the treated aqueous solutions can be discarded down the drain. If you have further questions
about EtBr please refer to the Material Safety Data Sheets (MSDS) provided by the vendor or manufacturer.

Note: Currently, the only acceptable alternative stain options are GelRedTM (Biotium, 31010), SYBR® Safe
(Invitrogen, S-33102) and SYBR® Gold (Invitrogen, S-11494). Labs are strongly encouraged to follow
manufacturer’s instructions and test stains in their labs before adopting them for routine use. If one of the
alternative stains is used, the destaining steps should be omitted.

2. Destain gel in approximately 500 ml CLRW for 60 - 90 min, changing water every 20 minutes. Capture image using
a Gel Doc 1000, 2000, EQ, XR, or equivalent documentation system. If too much background is observed destain
for an additional 30-60 min.

3. Follow directions given with the imaging equipment to save gel image as a *.1sc file; convert this file to *.tif file for
analysis with the BioNumerics software program. The gel image should fill the entire window of the imaging
equipment (computer) screen (without cutting off wells or lower bands). Ensure that the image is in focus and
that there is little to no saturation (over-exposure) in the bands (signified by red pixilation in the QuantityOne or
ImageLab software). Additional instructions are provided in PNL07 of the PulseNet QA/QC manual.

4. Drain buffer from electrophoresis chamber and discard. Rinse chamber with 2 L Ultrapure water (CLRW) or, if
unit is not going to be used for several days, flush lines with water by letting pump run for 5-10 min before draining
water from chamber and tubing.

5. If the lowest band in the H9812 standard does not migrate within 1-1.5 cm of the bottom of the gel, the proper run
time will need to be determined empirically for the conditions in each laboratory.

Note: The following options are available if PFGE results do not have to be available within 24-28 hours:

− Plugs can be lysed for longer periods of time (3-16 hours).

− The washing steps with TE to remove the lysis buffer from the PFGE plugs can be done for longer periods
of time (30-45 min) and at lower temperatures (37°C or room temperature). They can be started on Day 1
and finished on Day 2 after overnight refrigeration of the plugs in TE.

Use of trade names and commercial sources is for identification purposes only and does not imply endorsement by CDC
or the U.S. Department of Health and Human Services.

NOTE: CLIA LABORATORY PROCEDURE MANUAL REQUIREMENTS

Efforts have been made to assure that the procedures described in this protocol have been written in accordance with
the 1988 Clinical Laboratory Improvement Amendments (CLIA) requirements for a procedure manual (42 CFR
493.1211). However, due to the format required for training, the procedures will require some modifications and
additions to customize them for your particular laboratory operation.
Any questions regarding the CLIA requirements for a procedure manual, quality control, quality assurance, etc.,
should be directed to the agency or accreditation organization responsible for performing your laboratory's CLIA

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 12 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

inspection. In addition, some states and accreditation organizations may have more stringent requirements that will need
to be addressed.

Formulas of Selected Reagents used in PulseNet Standardized Laboratory Protocol for PFGE

Tris:EDTA Buffer, pH 8.0 (TE, 10 mM Tris:1 mM EDTA, pH 8.0)

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure water (CLRW)

Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosine + 0.1 mg/ml Proteinase K)
25 ml (50 ml) of 1 M Tris, pH 8.0
50 ml (100 ml) of 0.5 M EDTA, pH 8.0
50 ml (100 ml) 10% N-Lauroylsarcosine, Sodium salt (Sarcosyl)
OR
5 g (10 g) of N-Lauroylsarcosine, Sodium salt (Sarcosyl) 3
Dilute to 500 ml (1000 ml) with Sterile Ultrapure water (CLRW)

Add 25 μl Proteinase K stock solution (20 mg/ml) per 5 ml of cell lysis buffer just before use for a final
concentration in the lysis buffer of 0.1 mg/ml Proteinase K.

Use the following calculations for AvrII (BlnI) or SpeI (30 Units/plug slice):

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory
175 μl 1750 μl 2625 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl
BSA (10mg/ml) 2 μl 20 μl 30 μl
AvrII or BlnI (10 U/μl) 3 μl 30 μl 45 μl

Total Volume 200 μl 2000 μl 3000 μl

Note: Keep vial of restriction enzyme on ice or in insulated storage box (-20ºC) at all times.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:
8.1 In May 2007, two revisions were made to the Standardized PFGE Protocol for E. coli O157:H7,
Salmonella, and Shigella sonnei, including reduction in cell suspension concentrations and the removal of
3
If Sarcosyl powder is added directly to the other components of this reagent, warm the solution to 50-60ºC for 30-
60 minutes, or leave at room temperature for ≈2 hours to completely dissolve the Sarcosyl; adjust to the final
volume with sterile Ultrapure water (CLRW).
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 13 of 14
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF ESCHERICHIA CODE: PNL05
COLI O157:H7, ESCHERICHIA COLI NON-O157 (STEC), SALMONELLA SEROTYPES, Effective Date:
SHIGELLA SONNEIAND SHIGELLA FLEXNERI 03 24 2013

SDS from the plug agarose. These revisions were adopted following an extensive evaluation and validation
process which demonstrated that the revised protocol remained robust and reproducible in multiple
laboratories.
8.2 The phrase “Type I Water” has been changed to “Ultrapure Clinical Laboratory Reagent Water (CLRW).”
The water composition is the same, but this reflects a change in the terminology used by the Clinical
Laboratory Standards Institute (CLSI).
8.3 August 2010 this protocol was revised to include E. coli Non-O157 (STEC) and Shigella flexnei instructions.
8.4 August 2010 changes:
− Plug washing steps can be performed at 54-55 ºC rather than lowering to 50 ºC.
− References to specific restriction buffers have been removed.
− Moved reference to BSA out of footnotes and into the main text of the protocol.
− The word “Sterile” has been deleted in reference to diluting 5X or 10X TBE to 0.5X TBE. Non-sterile
CLRW is acceptable.
− Added a recommendation for laboratories to monitor the initial mAmps when electrophoresis is
started.
8.5. August 2010: A statement was included to allow the use of alternative agaroses for casting the running gel,
along with recommendations strongly urging each lab to optimize the run time. Internal and external
validation showed that run times could be affected by agarose type, but no trends were noted so a blanket
recommendation on run times cannot be made. Additional agarose alternatives may be tested and deemed
acceptable at a later date.
8.6. August 2010: A statement was included to allow the use of alternative DNA stains that are equivalent to
EtBr. Labs are strongly urged to follow manufacturer’s instructions as well as test stains in their own labs
to gain experience using alternative agarose stains. Additional stain alternatives may be tested and deemed
acceptable at a later date.
8.7. March 2013 changes:
− Corrected formula for TE buffer. TE used at CDC is 10 mM for Tris and 1 mM for EDTA.
− Recommended disinfectant changed from 10% bleach to 1% Lysol/Amphyll or 90% ethanol.
− Volume of TE needed for washing plugs was corrected from 300 – 350 ml to 400 – 600 ml.
− A statement was added to clarify that using combs with small teeth (5.5 mm) was not advised.
− Use of pre-restriction step and BSA was changed from optional to highly recommended. Calculation
for including BSA in restriction enzyme master mix was added.
− Statement allowing of Megbase agarose (BioRad) was deleted. Additional testing revealed run time
and normalization were negatively impacted by this agarose.
− Upper limit for starting mAmps was reduced from 170 to 150mAmps.
− The option to allow incubation times for restriction digestion to be increased longer than recommended
was deleted.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 14 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE AND Effective Date:
VIBRIO PARAHAEMOLYTICUS 03 24 2013

1. PURPOSE: To describe the One-Day (24-26 h) standardized laboratory protocol for molecular
subtyping of Vibrio cholerae and Vibrio parahaemolyticus by Pulsed-field Gel Electrophoresis
(PFGE).

2. SCOPE: To provide the PulseNet participants with a standardized procedure for performing PFGE of
Vibrio cholerae and Vibrio parahaemolyticus thus ensuring inter-laboratory comparability of the
generated results.

3. DEFINITIONS/TERMS:
3.1 PFGE: Pulsed-field Gel Electrophoresis
3.2 DNA: Deoxyribonucleic acid
3.3 CDC: Centers for Disease Control and Prevention
3.4 CLRW: Clinical Laboratory Reagent Water

4. RESPONSIBILITIES/PROCEDURE:
PREPARATION OF PFGE PLUGS FROM AGAR CULTURES

BIOSAFETY WARNING: Vibrio cholerae and Vibrio parahaemolyticus are human pathogens and can cause serious
disease. Always use Biosafety Level 2 practices and extreme caution when transferring and handling strains of these
genera. Work in a biological safety cabinet when handling large amounts of cells. Disinfect or dispose of all plasticware
and glassware that come in contact with the cultures in a safe manner.

Please read all instructions carefully before starting protocol. Treat all plasticware, glassware, pipets, spatulas, etc. that
come in contact with the cell suspensions or plugs as contaminated materials and dispose of, or disinfect according to the
guidelines of your institution. Disinfect reusable plug molds before they are washed; the disposable plug molds,
including the tape and the tab that is used to push the plugs out of the wells, are also contaminated and should be
disinfected with 1% Lysol/Amphyll or 90% ethanol for at least 30 minutes if they will be washed and reused.

Day 0
Streak an isolated colony from test cultures to Trypticase Soy Agar with 5% defibrinated sheep blood (TSA-SB) plates
(or comparable non-selective media) for confluent growth. It is recommended that a storage vial of each culture be
created. To do this, stab small screw cap tubes of Marine motility agar or similar medium with the same inoculating loop
used to streak the plate. This will ensure that the same colony can be retested if necessary Incubate cultures at 37ºC for
14-18 h.

Day 1
1. Turn on shaker water bath (54-55ºC), stationary water baths (55-60ºC) and spectrophotometer (or equivalent
instrument such as the Dade Microscan Turbidity meter or bioMérieux Vitek colorimeter).

2. Prepare TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0)1 as follows:

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure Clinical Laboratory Reagent Water (CLRW)

Note: The TE Buffer is used to make the plug agarose and also to wash lysed PFGE plugs.

1
Additional information is found on page 13 of this document.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE AND Effective Date:
VIBRIO PARAHAEMOLYTICUS 03 24 2013

3. Prepare 1% SeaKem Gold agarose in TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) for PFGE plugs as follows:
a. Weigh 0.50 g (or 0.25 g) SeaKem Gold (SKG) into 250 ml screw-cap flask.
b. Add 50 ml (or 25 ml) TE Buffer; swirl gently to disperse agarose.
c. Loosen or remove cap and cover loosely with clear film, and microwave for 30 sec; mix gently and repeat for 10
sec intervals until agarose is completely dissolved.
d. Recap flask and return to 55- 60ºC water bath and equilibrate the agarose for 15 minutes or until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: SeaKem Gold agarose works well for making PFGE plugs because it provides added strength to the plugs that are
cast in reusable plug molds, minimizing breakage of plugs during the lysis and washing steps. The time and temperature
needed to completely dissolve the agarose is dependent on the specifications of the microwave used, and will have to be
determined empirically in each laboratory.

4. Label small transparent tubes (12 mm x 75 mm Falcon 2054 tubes or equivalent) with culture numbers.

5. Prepare Cell Suspension Buffer (100 mM Tris:100 mM EDTA, pH 8.0) as follows:

10 ml of 1 M Tris, pH 8.0
20 ml of 0.5 M EDTA, pH 8.0
Dilute to 100 ml with sterile Ultrapure water (CLRW)

6. Transfer ≈2 ml of Cell Suspension Buffer (CSB) to small labeled tubes. Use a sterile polyester-fiber or cotton swab
that has been moistened with sterile CSB to remove some of the growth from agar plate; suspend cells in CSB by
spinning swab gently so cells will be evenly dispersed and formation of aerosols is minimized.

Note: The minimum volume of the cell suspension needed will depend on size of the cuvettes or tubes used to measure
the cell concentration and are dependent on the manufacturer’s specifications for the spectrophotometer, turbidity meter,
or colorimeter.

7. Adjust concentration of cell suspensions to one of values given below by diluting with sterile CSB or by adding
additional cells.
a. Spectrophotometer: 610 nm wavelength, absorbance (Optical Density) of 0.9 (range of 0.8-1.0)
b. Dade Microscan Turbidity Meter: 0.35 – 0.45 (measured in Falcon 2054 tubes)
0.52 – 0.64 (measured in Falcon 2057 tubes; V. cholerae)
0.55 – 0.65 (measured in Falcon 2057 tubes; V. parahaemolyticus)
c. bioMérieux Vitek colorimeter: ≈ 20% transmittance (measured in Falcon 2054 tubes)

Note: Cell suspensions need to be at room temperature when concentration is checked. The values in Steps 7a, 7b and
7c give satisfactory results at CDC; if different instruments or tubes are used, each laboratory may need to establish the
concentration needed for satisfactory results.

CASTING PLUGS

Label wells of PFGE plug molds with culture number. When reusable plug molds are used, put strip of tape on lower
part of reusable plug mold before labeling wells.

Note: Unused plug agarose can be kept at room temperature and reused 1-2 times. Microwave on low-medium power
for 10 -15 sec and mix; repeat for 5-10 sec intervals until agarose is completely melted. This agarose melts rapidly!

Note: Proteinase K solutions (20 mg/ml) are available commercially. Alternatively, a stock solution of Proteinase K can
be prepared from the powder in sterile Ultrapure water (CLRW). For best results, aliquot 300-500 μl into small tubes
VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 14
 STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE AND CODE: PNL06
Effective Date:
VIBRIO PARAHAEMOLYTICUS 03 24 2013

and store in a freezer (-20ºC) until ready to use. Just before use, thaw appropriate number of vials needed for the
samples; keep Proteinase K solutions on ice. If the Proteinase K stock solution was prepared from powder, discard any
thawed solution at the end of the work day. Store commercially prepared Proteinase K solutions according to directions
provided by the supplier.

1. Transfer 400 μl (0.4 ml) adjusted cell suspensions to labeled 1.5 ml microcentrifuge tubes.

2. Add 20 μl of Proteinase K (20 mg/ml stock) to each tube and mix gently with pipet tip. (200 μl is needed for 10 cell
suspensions.)

3. Add 400 μl (0.4 ml) melted 1% SeaKem Gold agarose to 400 µl cell suspension; mix by gently pipetting mixture up
and down a few times. Over-pipeting can cause DNA shearing. Maintain temperature of melted agarose by
keeping flask in beaker of warm water (55-60ºC).

4. Immediately, dispense part of mixture into appropriate well(s) of reusable plug mold. Do not allow bubbles to form.
Two plugs of each sample can be made from these amounts of cell suspension and agarose and are useful if repeat
testing is required. Allow plugs to solidify at room temperature for 10-15 min. They can also be placed in the
refrigerator (4ºC) for 5 minutes.

Note: If disposable plug molds are used for making plugs with 1% SeaKem Gold agarose, use 200 μl cell suspension,
10 μl of Proteinase K (20 mg/ml stock) and 200 μl of agarose; up to 4 plugs can be made from these amounts of cell
suspension and agarose.

Note: The generation of cell suspension and the subsequent casting of the plugs should be performed as rapidly as
possible in order to minimize premature cell lysis. If large numbers of samples are being prepared, it is recommended
that they be processed in batches of ~10 samples at a time. Once the first batch of isolates are in the cell lysis incubation,
then start preparing the cells suspensions the next group samples, and so on. All batches can be lysed and washed
together, since additional lysis time will not affect the initial batches.

LYSIS OF CELLS IN AGAROSE PLUGS

Note: Two plugs (reusable plug molds) or up to four plugs (disposable plug molds) of the same strain can be lysed in the
same 50 ml tube.

1. Label 50 ml polypropylene screw-cap or 50ml Oak Ridge tubes with culture numbers.

2. Prepare Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) as follows:
25 ml of 1 M Tris, pH 8.0
50 ml of 0.5 M EDTA, pH 8.0
50 ml of 10 % Sarcosyl (N-Lauroylsarcosine, Sodium salt) 2
Dilute to 500 ml with Ultrapure water (CLRW)

3. Calculate the total volume of Cell Lysis/Proteinase K Buffer needed as follows:

a. 5 ml Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) is needed per tube (e.
g., 5 ml x 10 tubes = 50 ml).

2
The N-Lauroylsarcosine, Sodium salt can be added directly to the other ingredients and allowed to dissolve. See
page 13 of this document.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

b. 25 μl Proteinase K stock solution (20 mg/ml) is needed per tube of the cell lysis buffer
(e. g., 25 μl x 10 tubes = 250 μl).
c. Prepare the master mix by measuring the correct volume of Cell Lysis Buffer and Proteinase K into appropriate
size test tube or flask and mix well.

Note: The final concentration of Proteinase K in the lysis buffer is 0.1 mg/ml, and is different from the concentration
that was added to the cell suspension (0.5 mg/ml).

4. Add 5 ml of Proteinase K/Cell Lysis Buffer to each labeled 50 ml tube.

5. Trim excess agarose from top of plugs with scalpel or razor blade (optional). Open reusable plug mold and transfer
plugs from mold with a 6-mm wide spatula to appropriately labeled tube. If disposable plug molds are used, remove
white tape from bottom of mold and push out plug(s) into appropriately labeled tube. Be sure plugs are under
buffer and not on side of tube.

Note: The excess agarose, plug mold, spatula, etc. are contaminated. Discard or disinfect appropriately.

6. Remove tape from reusable mold. Place both sections of the plug mold, spatulas, and scalpel in 90% ethanol, 1%
Lysol/Amphyll or other suitable disinfectant. Soak them for 15 minutes before washing them. Discard
disposable plug molds.

7. Place tubes in rack and incubate in a 54-55ºC shaker water bath or incubator for 1 hour with constant and vigorous
agitation (150-175 rpm). If lysing in water bath, be sure water level in water bath is above the level of lysis buffer in
tubes.

8. Pre-heat enough sterile Ultrapure water (CLRW) to 54-55ºC so that plugs can be washed two times with 10-15 ml
water (200-250 ml for 10 tubes).

WASHING OF AGAROSE PLUGS AFTER CELL LYSIS

Note: Most laboratories will find that their plugs are sufficiently stable to perform the following washing steps at
54-55ºC. However, if you notice that your plugs are nicked along the edges or breaking it will be necessary for your
laboratory to lower the water bath or incubator to 50ºC for the following washing steps.

1. Remove tubes from water bath, and carefully pour off lysis buffer into an appropriate discard container; plugs can be
held in tubes with a screened cap or spatula.

Note: It is important to remove all of the liquid during this and subsequent wash steps by touching edge of tube or
screened cap on an absorbent paper towel.

2. Add at 10-15 ml sterile Ultrapure water (CLRW) that has been pre-heated to 54-55ºC to each tube and shake the
tubes vigorously in a 54-55ºC water bath or incubator for 10-15 min.

3. Pour off water from the plugs and repeat wash step with pre-heated water (Step 2) one more time.
a. Pre-heat enough sterile TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) in a 54-55ºC water bath so that plugs can
be washed four times with 10-15 ml TE (400-600 ml for 10 tubes) after beginning last water wash.

4. Pour off water, add 10-15 ml pre-heated (54-55ºC) sterile TE Buffer, and shake the tubes vigorously in 54-55ºC
water bath or incubator for 10-15 min.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

5. Pour off TE and repeat wash step with pre-heated TE three more times.

6. Decant last wash and add 5-10 ml sterile TE. Continue with step 1 in "Restriction Digestion" section or store plugs
in TE Buffer at 4ºC until needed. Plugs can be transferred to smaller tubes for storage.

Note: If restriction digestion is to be done the same day, complete Steps 1-3 of next section (RESTRICTION
DIGESTION OF DNA IN AGAROSE PLUGS WITH SfiI or NotI) during last TE wash step for optimal use of time.

RESTRICTION DIGESTION OF DNA IN AGAROSE PLUGS

Note: A small slice of the plug (not the entire plug) should be digested with the primary restriction enzyme because less
enzyme is required and other slices of the plug can be subjected to restriction analysis with other enzymes. SfiI is
recommended as the secondary enzyme for analysis of Vibrio cholerae and Vibrio parahaemolyticus. The use of a
secondary enzyme is useful in situations where the PFGE patterns obtained with the primary enzyme from two or more
isolates are indistinguishable.

1. Label 1.5 ml microcentrifuge tubes with culture numbers; label 3 (10-well gel) or 4 (15-well gel) tubes for
Salmonella ser. Braenderup H9812 3 standards.

a. Pre-Restriction Incubation Step (highly recommended): Prepare a master mix by diluting the appropriate
10X restriction buffer (Roche Applied Science or equivalent) 1:10 with sterile Ultrapure water (CLRW)
according to the following table:

Note: The appropriate restriction buffer will vary between vendors and may differ between enzymes from the same
vendor. Always use the restriction buffer recommended by the vendor for the particular restriction enzyme.

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory

180 μl 1800 μl 2700 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl

Total Volume 200 μl 2000 μl 3000 μl

b. Add 200 μl diluted restriction buffer (1X) to labeled 1.5 ml microcentrifuge tubes.
c. Carefully remove plug from TE with spatula and place in a sterile disposable Petri dish or on large glass slide.
d. Cut a 2.0 to 2.5 mm-wide slice from test samples with a single edge razor blade (or scalpel, cover slip, etc.) and
transfer to tube containing diluted restriction buffer. Be sure plug slice is under buffer. Replace rest of plug
in original tube that contains 5 ml TE buffer and store at 4ºC.

Note: PulseNet recommends that the combs with larger teeth (10 mm-wide teeth) be used to cast the gels because
computer analysis of the gel lanes is more accurate and less tedious than analysis of gel lanes cast with combs with the
smaller teeth (5.5 mm). Using combs with smaller teeth is not advised. The number of slices that can be cut from the
plugs will depend on the skill and experience of the operator, integrity of the plug, and whether the slices are cut
vertically or horizontally (plugs made in disposable molds).

3
Directions for making and testing PFGE plugs of Salmonella ser. Braenderup H9812 are in PNL05.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

e. Incubate sample and control plug slices in water bath or incubator for 5-10 min or at room temperature for 10-15
min.
i. Incubate samples to be restricted with SfiI at 50°C.
ii. Incubate samples to be restricted with NotI and XbaI at 37ºC.
f. After incubation, remove buffer from plug slice using a pipet fitted with 200-250 μl tip all the way to bottom of
tube and aspirate buffer. Be careful not to cut plug slice with pipet tip and that plug slice is not discarded with
pipet tip.

2. Prepare the restriction enzyme master mix according to the following table. May mix in the same tube that was used
for the diluted restriction buffer:

Note: Enzymes may be purchased in several different stock concentrations. The SfiI stock enzyme should be ordered in
concentrated form (40 U/µl) rather than unconcentrated form (10 U/µl). Either form is acceptable for NotI restriction.
The calculations below are based on using an enzyme at a concentration of 40 U/μl. If a different concentration of
enzyme is used, make necessary adjustments to the volume of enzyme and water to achieve a final concentration of 40
U/ sample.

Note: Keep vial of restriction enzyme on ice or in insulated storage box (-20˚C) at all times.

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory
177μl 1777 μl 2655 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl
BSA (10mg/ml) 2 μl 20 μl 30 μl
SfiI (40 U/μl) 1μl 10 μl 15 μl

Total Volume 200 μl 2000 μl 3000 μl

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory
177 μl 1777 μl 2655 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl
BSA (10mg/ml) 2 μl 20 μl 30 μl
NotI (40 U/μl) 1 μl 10 μl 15 μl

Total Volume 200 μl 2000 μl 3000 μl

Note: Addition of Bovine Serum Albumin (BSA; highly recommended): Several restriction enzyme vendors
specifically recommend the addition of 1X BSA to enzyme restriction mixtures while others do not. PulseNet
Central recommends adding BSA to all enzyme restriction mixtures to minimize the incidence of incomplete
restriction.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

3. Add 200 μl restriction enzyme master mix to each tube. Close tube and mix by tapping gently; confirm that plug
slices are under enzyme mixture.

4. Incubate sample and standard (control) plug slices for 4 hours in a water bath at the appropriate temperature for
the enzyme.
a. Incubate samples restricted with SfiI at 50°C
b. Incubate samples restricted with NotI and XbaI at 37°C.

5. If plug slices will be loaded into the wells (Option B, page 9), continue with Steps 1-4 of the next section
(CASTING AGAROSE GEL) approximately 1 h before restriction digest reaction is finished so the gel can
solidify for at least 30 minutes before loading the restricted PFGE plugs.

CASTING AGAROSE GEL

A. Loading Restricted Plug Slices on the Comb:

1. Confirm that water bath is equilibrated to 55 - 60ºC.

2. Make volume of 0.5X Tris-Borate EDTA Buffer (TBE) that is needed for both the gel and electrophoresis running
buffer according to one of the following tables.

5X TBE:
Reagent Volume in milliliters (ml)
5X TBE 200 220
Clinical Laboratory Reagent Water
1800 1980
(CLRW)
Total Volume of 0.5X TBE 2000 2200

10X TBE:
Reagent Volume in milliliters (ml)
10X TBE 100 110
Clinical Laboratory Reagent
1900 2090
Water (CLRW)
Total Volume of 0.5X TBE 2000 2200

3. Make 1% SeaKem Gold (SKG) Agarose in 0.5X TBE as follows:

a. Weigh appropriate amount of SKG into 500 ml screw-cap flask.
b. Add appropriate amount of 0.5X TBE; swirl gently to disperse agarose.
i. Mix 1.0 g agarose with 100 ml 0.5X TBE for 14 cm-wide gel form (10 wells)
ii. Mix 1.5 g agarose with 150 ml 0.5X TBE for 21 cm-wide gel form (15 wells)
c. Remove cap or cover loosely with clear film, and microwave for 60 sec; mix gently and repeat for 15 sec
intervals until agarose is completely dissolved.
d. Recap flask and return to 55- 60ºC water bath and equilibrate the agarose in the water bath for 15 minutes or
until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

Note: Agarose LFTM (Amresco, X174) is the only acceptable alternative to SeaKem Gold at this time. The time and
temperature needed to completely dissolve the agarose is dependent on the specifications of the microwave used and will
have to be determined empirically in each laboratory. Similarly, the optimal running time for each agarose will have to
be determined empirically in each laboratory.

4. A small volume (2-5 ml) of melted and cooled (50-60ºC) 1% SKG agarose may be wanted to seal wells after plugs
are loaded. Prepare as described above. Unused SKG agarose can be kept at room temperature, melted, and
reused several times.

Note: Place the gel form on a leveling table and adjust until perfectly leveled. Place the comb holder so the front part
(side with small metal screws) and teeth face the bottom of gel frame and the comb teeth touch the gel platform.

5. Remove restricted plug slices from 50°C or 37ºC water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X
TBE. Incubate at room temperature for 5 min.

6. Remove plug slices from tubes; put comb on bench top and load plug slices on the bottom of the comb teeth as
follows:
a. Load S. ser. Braenderup H9812 standards on teeth (lanes) 1, 5, 10 (10-well gel) or on teeth 1, 5, 10, 15 (15-well
gel).
b. Load samples on remaining teeth and note locations.

7. Remove excess buffer with tissue or kimwipe. Allow plug slices to air dry on the comb for 5-10 minutes or seal them
to the comb with 1% SKG agarose (55-60ºC).

8. Position comb in leveled gel form and confirm that the plugs slices are correctly aligned on the bottom of the comb
teeth, and that the lower edge of the plug slice is flush against the black platform.

9. Carefully pour the agarose (cooled to 55-60ºC) into the gel form and remove any bubbles or debris.

10. Put black gel frame in electrophoresis chamber. Add 2 -2.2 L freshly prepared 0.5X TBE. Close cover of unit. (The
amount of buffer needed depends on whether residual buffer was left in tubing or if unit was flushed with water
after the last gel was run.)

11. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute (setting of ~70) and cooling module (14ºC)
approximately 30 minutes before gel is to be run.

12. Remove comb after gel solidifies, about 30-45 minutes.

13. Fill in wells of gel with melted and cooled (55- 60ºC) 1% SKG Agarose (optional). Unscrew and remove end gates
from gel form; remove excess agarose from sides and bottom of casting platform with a tissue or kimwipe. Keep
gel on casting platform and carefully place gel inside black gel frame in electrophoresis chamber. Close cover of
chamber.

B. Loading Restricted Plug Slices into the Wells:

1. Follow steps 1-4 in Section A on pages 7 and 8 (Loading Restricted Plug Slices on the Comb).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

Note: Place the gel form on a leveling table and adjust until perfectly leveled before pouring gel. Position the comb
holder so that the front part (side with small metal screws) and teeth face the bottom and the bottom of the comb is 2 mm
above the surface of the gel platform.

2. Cool melted SKG agarose in 55-60ºC water bath for 15-20 min; carefully pour agarose into gel form (casting stand)
fitted with comb. Be sure there are no bubbles.

3. Put black gel frame in electrophoresis chamber. Add 2-2.2 L freshly prepared 0.5X TBE. Close cover of unit. (The
amount of buffer depends on whether residual buffer was left in tubing, or if unit was flushed with water after the
last gel was run).

4. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute (setting of ~70) and cooling module (14ºC)
approximately 30 minutes before gel is to be run.

5. Remove restricted plug slices from water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE. Incubate
at room temperature for 5 minutes.

6. Remove comb after gel solidifies, about 30 – 45 minutes.

7. Remove restricted plug slices from tubes with tapered end of spatula and load into appropriate wells. Gently push
plugs to bottom and front of wells with wide end of spatula. Manipulate position with spatula and be sure that are
no bubbles.
a. Load S. ser. Braenderup H9812 standards in wells (lanes) 1, 5, 10 (10-well gel) or in wells 1, 5, 10, 15 (15-well
gel).
b. Load samples in remaining wells.

Note: Loading the plug slices can be tedious; each person has to develop his/her own technique for consistently placing
the plug slices in the wells so the lanes will be straight and the bands sharp.

8. Fill in wells of gel with melted 1% SKG Agarose (equilibrated to 55-60ºC). Allow to harden for 3-5 min. Unscrew
and remove end gates from gel form; remove excess agarose from sides and bottom of casting platform with a tissue
or kimwipe. Keep gel on casting platform and carefully place gel inside black gel frame in electrophoresis chamber.
Close cover of chamber.

ELECTROPHORESIS CONDITIONS

1. Select following conditions for V. cholerae strains restricted with SfiI and NotI :
a. Select following conditions on the CHEF Mapper with a two-block program
Block 1: 2 s - 10 s, 13 hours
Block 2: 20 s - 25 s, 6 hours
1. Press the Multi-State button on the Chef Mapper.
2. Program with Interrupts?
0 = No
Note: Press ‘Enter’ after each value or command is entered.
3. Block 1 Runtime?
13 hours
4. Block 1, State 1: (Fill in the blanks appropriately)
a. 6.0 volts
b. angle = 60.0
c. Initial switch time = 2 s
VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

d. Final switch time = 10 s

e. Ramping factor, a = 0 (linear)
5. Continue with another state (Vector)?
1 = Yes
6. Block 1, State 2: (Fill in the blanks appropriately)
a. 6.0 volts
b. angle = - 60.0
Note: The angle for State 2 is Negative
c. Initial switch time = 2 s
d. Final switch time = 10 s
e. Ramping factor, a = 0 (linear)
7. Continue with another state (Vector)?
0 = No
8. Continue with another Block?
1 = Yes
9. Block 2 Runtime?
6 hours
10. Block 2, State 1: (Fill in the blanks appropriately)
a. 6.0 volts
b. angle = 60.0
c. Initial switch time = 20 s
d. Final switch time = 25 s
e. Ramping factor, a = 0 (linear)
11. Continue with another state (Vector)? 1 = Yes
12. Block 2, State 2: fill in the blanks appropriately.
a. 6.0 volts
b. angle = - 60.0
Note: The angle for State 2 is Negative
c. Initial switch time = 20 s
d. Final switch time = 25 s
e. Ramping factor, a = 0 (linear)
13. Continue with another state (Vector)? 0 = No
14. Continue with another Block? 0 = No
15. A program is in memory, please enter another command.
16. Press the Start Run Button

b. Select the following conditions on CHEF DR-III

Block I:
Initial switch time: 2s
Final Switch time: 10s
Voltage: 6V
Included Angle: 120°
Run time: 13 h
Block II:
Initial switch time: 20s
Final switch time: 25s
Voltage: 6V
Included Angle: 120°
Run time: 6 h

2. Select following conditions for V. parahaemolyticus strains restricted with SfiI and NotI :
a. Select following conditions on the CHEF Mapper

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

Auto Algorithm
78 kb - low MW
396 kb - high MW
Select default values except where noted by pressing "enter.”
Change run time to 18 – 19 hr (See note below)
(Default values: Initial switch time = 10 s; Final switch time = 35.03 s)
Linear ramping factor

b. Select the following conditions on CHEF DR-III

Initial switch time: 10 s
Final switch time: 35 s
Voltage: 6 V
Included Angle: 120°
Run time: 18 - 19 hr

c.Select the following conditions on CHEF DR-II

Initial A time: 10 s
Final A time: 35 s
Start Ragio: 1.0 (if applicable)
Voltage: 200 V
Run time: 18 - 19 hr

Note: The electrophoresis running times recommended above are based on the equipment and reagents used at the CDC.
Run times may be different in your laboratory and will have to be optimized for your gels so that the lowest band
in the S. ser. Braenderup H9812 standard migrates 1.0 - 1.5 cm from the bottom of the gel.

Note: Make note of the initial milliamp (mAmp) reading on the instrument. The initial mAmps should be between 110-
150 mAmps. A reading outside of this range may indicate that the 0.5X TBE buffer was prepared improperly and the
buffer should be remade.

Day 2

STAINING AND DOCUMENTATION OF PFGE AGAROSE GEL

Note: The following staining procedure describes the use of ethidium bromide to stain PFGE gels. Alternate DNA
stains may be used. Please see the “Alternate DNA Stains-Results and Recommendations” posting within the PulseNet
Documents forum on the SharePoint site for additional information.

1. When electrophoresis run is over, turn off equipment; remove and stain gel with ethidium bromide by diluting40 μl
of ethidium bromide stock solution (10 mg/ml) with 400 ml of Ultrapure water (CLRW). This volume is for a
staining box that is approximately 14 cm x 24 cm; a larger container may require a larger amount of staining
solution. Stain gel for 20-30 min in covered container.

Note: Ethidium bromide is toxic and a mutagen. Stock solutions of 10 mg/ml Ethidium Bromide (EtBr) in water are
available from several commercial companies (Amresco X328; Bio-Rad, 161-0433; Sigma, E-1510). The diluted
solution can be kept in dark bottle and reused 6-8 times before discarding according to your institution's guidelines
for hazardous waste. CDC does not recommend disposing of EtBr down the drain. Aqueous solutions containing
EtBr can be filtered through charcoal or degraded using activated carbon destaining or “tea” bags from Amresco
(E732-25 Destaining Bags) or other companies, which effectively and safely remove EtBr from solutions and gels.
Once the EtBr is removed, the treated aqueous solutions can be discarded down the drain. If you have further
VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

questions about EtBr please refer to the Material Safety Data Sheets (MSDS) provided by the vendor or
manufacturer.

Note: Currently, the only acceptable alternative stain options are GelRedTM (Biotium, 31010), SYBR® Safe
(Invitrogen, S-33102) and SYBR® Gold (Invitrogen, S-11494). Labs are strongly encouraged to follow
manufacturer’s instructions and test stains in their labs before adopting them for routine use. If one of the
alternative stains is used, the destaining steps should be omitted.

2. Destain gel in approximately 500 ml Ultrapure water (CLRW) for 60 - 90 min, changing water every 20 minutes.
Capture image on a Gel Doc 1000, Gel Doc 2000, or equivalent documentation system. If background interferes
with resolution, destain for an additional 30-60 min.

3. Follow directions given with the imaging equipment to save gel image as an *.1sc file; convert this file to *.tif file for
analysis with the BioNumerics software program. The gel image should fill the entire window of the imaging
equipment (computer) screen (without cutting off wells or lower bands). Ensure that the image is in focus and
that there is little to no saturation (over-exposure) in the bands (signified by red pixilation in the QuantityOne or
ImageLab software). Additional instructions are provided in PNL07 of the PulseNet QA/QC manual.

4. Drain buffer from electrophoresis chamber and discard. Rinse chamber with 2 L Ultrapure water (CLRW) or, if unit
is not going to be used for several days, flush lines with water by letting pump run for 5-10 min before draining
water from chamber and tubing.

5. If the lowest band in the H9812 standard does not migrate within 1-1.5 cm of the bottom of the gel, the proper run
time will need to be determined empirically for the conditions in each laboratory.

Note: The following options are available if PFGE results do not have to be available within 24-28 hours:
− Plugs can be lysed for longer periods of time (5-16 hours).
− The washing steps with TE to remove the lysis buffer from the PFGE plugs can be done for longer periods of
time (30-45 min) and at lower temperatures (37°C or room temperature). They can be started on Day 1
and finished on Day 2 after overnight refrigeration of the plugs in TE.

Use of trade names and commercial sources is for identification purposes only and does not imply endorsement by CDC
or the U.S. Department of Health and Human Services.

NOTE: CLIA LABORATORY PROCEDURE MANUAL REQUIREMENTS

Efforts have been made to assure that the procedures described in this protocol have been written in accordance with
the 1988 Clinical Laboratory Improvement Amendments (CLIA) requirements for a procedure manual (42 CFR
493.1211). However, due to the format required for training, the procedures will require some modifications and
additions to customize them for your particular laboratory operation.
Any questions regarding the CLIA requirements for a procedure manual, quality control, quality assurance, etc.,
should be directed to the agency or accreditation organization responsible for performing your laboratory's CLIA
inspection. In addition, some states and accreditation organizations may have more stringent requirements that will need
to be addressed.

Formulas of Selected Reagents used in PulseNet Standardized Laboratory Protocol for PFGE

VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

Tris:EDTA Buffer, pH 8.0 (TE, 10 mM Tris:1 mM EDTA, pH 8.0) 4

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure water (CLRW)

Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosine + 0.1 mg/ml
Proteinase K)
25 ml (50 ml) of 1 M Tris, pH 8.0
50 ml (100 ml) of 0.5 M EDTA, pH 8.0
50 ml (100 ml) 10% N-Lauroylsarcosine, Sodium salt (Sarcosyl)
OR
5 g (10 g) of N-Lauroylsarcosine, Sodium salt (Sarcosyl) 5
Dilute to 500 ml (1000 ml) with sterile Ultrapure water (CLRW)

Add 25 μl Proteinase K stock solution (20 mg/ml) per 5 ml of cell lysis buffer just before use for a final concentration in
the lysis buffer of 0.1 mg/ml Proteinase K.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:
8.1. The phrase “Type I Water” has been changed to “Ultrapure Clinical Laboratory Reagent Water
(CLRW).” The water composition is the same, but this reflects a change in the terminology used by
the Clinical Laboratory Standards Institute (CLSI).
8.2. March 2013 changes:
− Protocol was revised to combine Vibrio cholerae and parahaemolyticus instructions.
− Corrected formula for TE buffer. TE used at CDC is 10mM for Tris and 1 mM for EDTA.
− Recommended disinfectant changed from 10% bleach to 1% Lysol/Amphyll or 90% ethanol.
− Corrected 1% SKG / TE plug agarose recipe.
− A note was added to provide guidance when working with large numbers of isolates (>10).
− Plug washing steps can be performed at 54-55ºC rather than lowering to 50ºC.
− Volume of TE needed to wash 10 plugs was corrected from 300 – 350 ml to 400 – 600 ml.
− A statement was added to clarify that using combs with small teeth (5.5 mm) was not advised.
− References to specific restriction buffers have been removed.
− Moved reference to BSA out of footnotes and into the main text of the protocol. Use of pre-
restriction step and BSA was changed from optional to highly recommended. Calculation for
including BSA in restriction enzyme master mix was added.

4
TE Buffer used at CDC is 10 mM for Tris and 1mM for EDTA
5
If Sarcosyl powder is added directly to the other components of this reagent, warm the solution to 50- 60ºC
for 30-60 minutes, or leave at room temperature for ≈2 hours to completely dissolve the Sarcosyl; adjust to the
final volume with sterile Ultrapure Water.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 14
 CODE: PNL06
STANDARD OPERATING PROCEDURE FOR PFGE OF VIBRIO CHOLERAE Effective Date:
03 24 2013

− The word “Sterile” has been deleted in reference to diluting 5X or 10X TBE to 0.5X TBE.
Non-sterile CLRW is acceptable.
− A statement was included to allow the use of an alternative agarose for casting the running gel,
along with recommendations strongly urging each lab to optimize the run time. Internal and
external validation showed that run times could be affected by agarose type, but no trends
were noted so a blanket recommendation on run times cannot be made. Additional agarose
alternatives may be tested and deemed acceptable at a later date.
− Added parameters for programing a CHEF DR-II for Vibrio parahaemolyticus.
− Added a recommendation for laboratories to monitor the initial mAmps when electrophoresis is
started.
− A statement was included to allow the use of alternative DNA stains that are equivalent to EtBr.
Labs are strongly urged to follow manufacturer’s instructions as well as test stains in their
own labs to gain experience using alternative agarose stains. Additional stain alternatives
may be tested and deemed acceptable at a later date.
− The option to allow incubation times for restriction digestion to be increased longer than
recommended was deleted.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 14 of 14
 STANDARD OPERATING PROCEDURE FOR IMAGE ACQUISITION AND CODE: PNL07
Effective Date:
PRODUCTION OF TIFF FILES FOR DATA ANALYSIS 07 26 2011

1. PURPOSE: To establish guidelines for the standardization of gel image acquisition using the GelDoc 1000,
2000, EQ, or XR Systems; and production of TIFF files for data analysis.

2. SCOPE: This procedure applies to all gels which will be analyzed using the BioNumerics software.

3. DEFINITIONS/TERMS:

3.1. SOP: Standard Operating Procedure

3.2. DI: distilled water

4. RESPONSIBILITIES:

5. PROCEDURE:

5.1. GelDoc 1000

5.1.1. After adequate staining and destaining of the agarose gel, a TIFF image of the gel is required for
analysis using the BioNumerics software. Handle the gel with untreated gloves (no powder or aloe)
at all times; remove or change gloves when using the computer keyboard or mouse.
5.1.2. Turn on the computer and printer connected with the GelDoc 1000 system. Open the Windows
directory. Using the mouse, double click on the Molecular Analyst icon. Click on File → New. The
default for the View window is Live.
5.1.3. Open the door of the GelDoc 1000 and carefully remove the gel from the appropriate container with
gloved hands or gel scoop; drain excess liquid from gel and place on the transilluminator platform
inside the GelDoc 1000. Use the black gel frame (with the pegs removed) to help prevent the gel
from sliding on the transilluminator platform. Turn on the white light using the switch on the upper
right hand corner of the GelDoc 1000. Using the computer monitor to visualize the gel, center the
gel on screen with the wells parallel to the top of the screen so that the wells are still visible. The
camera aperture (F stop) should be barely open (setting of 2-3) to allow for proper exposure of the
gel (adjustment is done with the top ring on the camera).
5.1.4. Make sure that the image completely fills the window and includes the wells on the top of the
screen. Slowly turn the middle ring of the camera to zoom in (or out) as close as possible to
eliminate blank space around the top, bottom and sides of the gel, but do not cut off blank wells or
the bottom of the gel. Using a flat ruler or grid, focus the image until it is sharp by turning the
bottom ring of the camera. If necessary, once the image is in focus make minor adjustments by
zooming in or out to ensure that the image size is appropriate. Minor adjustments to the image size
should not change the focus. Once the GelDoc is focused properly, very little adjustment should be
needed for future gels. Remove gloves for computer operation. Close the door, turn off the white
light switch, and turn on the UV light which is located at the lower right hand corner of the GelDoc.
(Labeled Power). NOTE: The UV light will not come on if the door is ajar or open. Be sure that the
toggle switch on the UV light box is set for “Analytical” and not “Preparative.”
5.1.5. It is common for the Live (Initial) image to be dark. Select Integrate from the View window to
visualize the image. Adjust the Integration Time until a satisfactory image is obtained. Bands on
every lane should be visible without excessive brightness. NOTE: Optimize the Integration Time by
selecting Show Saturation and adjusting the integration time by turning the top ring of the camera so
that the strongest sample band (DNA) is just below the point of saturation (no red showing).
Saturation in the gel wells may be present and is acceptable. If the image is not visible, increase the
integration times or check the aperture on the camera (top ring). Adjust the aperture to the
appropriate level of brightness by opening it up to the maximum setting. If the image is still not
visible, the gel may have to be restained with ethidium bromide.
5.1.6. Once the desired image has been captured, select Freeze from the View window and turn off the UV
light to avoid quenching the DNA in the gel.
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 1 of 5
 STANDARD OPERATING PROCEDURE FOR IMAGE ACQUISITION AND CODE: PNL07
Effective Date:
PRODUCTION OF TIFF FILES FOR DATA ANALYSIS 07 26 2011

5.1.7. To save captured image, select File → Save and name it using the .img extension and click OK. Use
the modify View icon to increase or decrease the brightness of the image by clicking on the right and
left arrows. When the adjustment is satisfactory, the Tools menu can be used to add a boxed text to
the gel image (optional). Once a satisfactory image has been acquired, select View → Show Full
Screen and print image.
5.1.8. To create a TIFF file, select File →Save As and change the extension to “.tif” and click OK. This
file can be either saved to your local computer (i.e. C drive) or to an external source (i.e. disk or CD).
5.1.9. Once the image has been successfully captured, remove the gel and place it in DI water until analysis
is completed. Thoroughly clean the transilluminator platform with DI water and/or 70% Isopropanol
and a soft, lint free towel (Kay-Dry or equivalent) making sure not to scratch the surface

5.2. GelDoc 2000 / Molecular Analyst Software or Quantity One

5.2.1. After adequate staining and de-staining of the agarose gel, a TIFF image of the gel is required for
analysis using the BioNumerics software. Handle the gel with untreated gloves (no powder or aloe)
at all times; remove or change gloves when using the computer keyboard or mouse.
5.2.2. Turn on the computer and printer connected with the GelDoc 2000 system. Open the Windows
directory. Double click on the Quantity One icon with the mouse. Click on File→Gel Doc. The
default for the View window is Live.
5.2.3. Open the drawer of the GelDoc 2000 and carefully remove gel from container with gloved hands or
gel scoop. Drain excess liquid from gel and place on the transilluminator platform. Use the black gel
frame (with the pegs removed) to help prevent the gel from sliding on the transilluminator platform.
Close drawer and open door; turn on the white light switch (Epi-Light). Using the computer monitor
to visualize the gel, center the gel on screen with the wells parallel to the top of the screen so that the
wells are still visible. The camera aperture (F stop) should be almost open: a setting of 2-3 on the
aperture will allow for proper exposure (adjustment is done with the top ring on the camera). Check
the box on the screen for the alignment grid so it can be used to help align the gel.
5.2.4. Make sure that the image completely fills the window and includes the wells on the top of the
screen. Slowly turn the middle ring of the camera to zoom in (or out) as close as possible to
eliminate blank space around the top, bottom and sides of the gel, but do not cut off blank wells or
the bottom of the gel. Using a flat ruler or grid, focus the image until it is sharp by turning the
bottom ring of the camera. If necessary, once the image is in focus make minor adjustments by
zooming in or out to ensure that the image size is appropriate. Minor adjustments to the image size
should not change the focus. Once the GelDoc is focused properly, very little adjustment should be
needed for future gels. Remove gloves for computer operation. Close the door, turn off the Epi-
Light switch, and turn on the Transilluminator (UV light). NOTE: The UV light will not come on if
the door is ajar or open.
5.2.5. It is common for the Live (Initial) image to be dark. Select Auto-Expose to determine an
approximate exposure time. The exposure can be “fine tuned” using Manual Expose and clicking on
the up and down arrows. For optimal analysis of the gel image, there should be little to no saturation
in the bands. Check Highlight Saturated Pixels to determine the level of saturation, and adjust the
amount of saturation by clicking the arrow icons and/or changing the camera aperture (top ring) so
that the strongest sample band (DNA) is just below the point of saturation (no red showing).
Saturation in the gel wells may be present and is acceptable. Inverted images can also be assessed in
real time by clicking the Invert Display. Refer to the manual for other options for the display or
annotation of the gel. NOTE: Exposure Time is equivalent to Integration Time as seen in previous
GelDoc systems.
5.2.6. Once the desired image has been captured, select Freeze from the View window and turn off the UV
light to avoid quenching the DNA in the gel.
5.2.7. A picture of the image (or inverted image) can be taken anytime by clicking Video Print. This also
freezes the image.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 2 of 5
 STANDARD OPERATING PROCEDURE FOR IMAGE ACQUISITION AND CODE: PNL07
Effective Date:
PRODUCTION OF TIFF FILES FOR DATA ANALYSIS 07 26 2011

5.2.8. To save the image, click File→Save. A name will be suggested corresponding to the date and time
of the image with a .1sc file extension. Change the filename to the appropriate designation, i.e.,
CDC11001.1sc. Print the saved image by clicking File→Print→Video Print.
5.2.9. To create a TIFF file, open the saved file and select File → Export to Tiff Image→Export and save
in appropriate drive. The file will have a corresponding name with the “.tif” extension.
5.2.10. Once the image has been successfully captured, remove the gel and place it in DI water until analysis
is completed. Use soft, lint-free towels (Kay-Dry or equivalent) to remove excess water from surface
of transilluminator and clean using DI water or 70% isopropanol making sure not to scratch the
surface.

5.3. GelDoc EQ and XR (Quantity One Software – version 4.5.0 or higher).

5.3.1. After adequate staining and de-staining of the agarose gel, a TIFF image of the gel is required for
analysis using the BioNumerics software. Handle the gel with untreated gloves (no powder or aloe)
at all times; remove or change gloves when using the computer keyboard or mouse.
5.3.2. Turn on Universal Hood II main switch (if off); turn on the computer and printer connected with the
GelDoc EQ or XR system. On the Windows desktop, double click on the Quantity One icon. Click
on File–>Gel Doc EQ or Gel Doc XR. The default for the View window is Live/Focus. (If
“Volumes Quick Guide” box appears in upper right of screen, it can be closed by clicking the “x” in
upper right hand corner of box.)
5.3.3. Open the drawer of the Universal Hood II of the GelDoc EQ or XR and carefully remove gel from
container with gloved hands or gel scoop. Drain excess liquid from gel, and place it on the
transilluminator platform. Use the black gel frame (with the pegs removed) to help prevent the gel
from sliding on the transilluminator platform. Close drawer and open door of the hood. Press the Epi-
Illumination key on the Light Source membrane touch pad control panel (upper) to turn on the Epi
White lights. While watching the computer screen, position the gel so that it is centered and the wells
are visible and parallel to the top of the screen. Check the box Show Alignment Grid to help align
the gel. Using the Lens Control touch pad (lower) on the cabinet or the Iris (camera aperture),
Zoom, or Focus arrows on the left side of the computer screen (Step I box), adjust the Iris so the gel
image is light – medium gray and the Zoom so that the gel image completely fills the window
without cutting off the wells, the bottom of the gel, or any blank wells. Place a clean, flat ruler on
the top of the gel and adjust the focus until the letters or numbers are sharp and clear on the computer
image; a “gel-cutter ruler” from Bio-Rad can also be used alone or placed beside the gel to help
adjust the focus.
5.3.4. Once the image is focused properly, very little adjustment should be needed for future gels. Close
the door, turn off the Epi White light and turn on the Trans UV light by pressing the keys of the
Light Source touch pad on the Universal Hood II. Be sure the UV button is selected in the Image
Mode (Step II). NOTE: The UV light will not come on if the drawer or door is ajar or open.
5.3.5. It is common for the Live/Focus image to be dark. Check Highlight Saturated Pixels (Step IV box).
Select Auto-Expose (Step III box) and then make any minor adjustments to the size of the gel image
with the Zoom arrow or touch pad key; use the Iris arrow or touch pad key so that the strongest
sample band (DNA) is just below the point of saturation (no red showing). Saturation in the gel wells
may be present and is acceptable; however, there should be no red color or saturation in any bands of
the PFGE patterns. The exposure time can also be adjusted by clicking on Manual Expose and using
the arrows below to adjust the iris (camera aperture) so that all saturation is removed. For most Gel
Doc systems, the default image is white bands on a dark background; if dark bands on a white
background (inverted image) are preferred, check the Invert Display (Step IV box). Refer to the
manual for other display or analysis options and annotation of the gel images (Step V). NOTE:
Exposure Time is equivalent to Integration Time as seen in previous GelDoc systems.
5.3.6. Once a satisfactory image of the gel is obtained, click Freeze (Step III). Turn off the Trans UV light
on the touch pad to avoid quenching the DNA in the gel.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 3 of 5
 STANDARD OPERATING PROCEDURE FOR IMAGE ACQUISITION AND CODE: PNL07
Effective Date:
PRODUCTION OF TIFF FILES FOR DATA ANALYSIS 07 26 2011

5.3.7. A picture of the image (or inverted image) can be taken anytime by clicking Video Print (Step VI).
This also freezes the image. Check a print of the gel image before saving it to make sure that it is
satisfactory and does not need any adjustments to the gel position or focus.
5.3.8. Save the image by clicking Save under Step VI (Select Output). A name will be suggested
corresponding to the date and time of the Raw 1-D image (*.1sc file extension). The filename should
be changed to the appropriate designation, i.e., CDC11001.1sc.
5.3.9. To create a TIFF file on the GelDoc EQ, open the saved image, select File → Export to Tiff Image.
Check Export in lower box and export the file to your local computer (i.e. C drive) or to an external
source (i.e. disk or flash drive). It will have a corresponding name with the “.tif” extension.
5.3.9.1. The camera in the GelDoc XR has much higher resolution than older models of the GelDoc
systems; thus, the file size is much larger (~1MB) compared to the size of the files generated
by cameras with lower resolution (~300Kb). Although the larger images can be analyzed in
BioNumerics, it is not ideal for storage either at CDC or in local databases. There are
limitations to the amount of data that can be stored at CDC and the tripling of file sizes would
not be manageable. The default settings that are set up when the PulseNet scripts are installed
(thickness of strips, resolution, etc.) are set for images that are ~300-400 Kb. After the image
acquisition step is completed, the file size of the image can be reduced using the following
instructions: select File → Export to Tiff Image. At this screen, Under Export Mode,
Publishing, select Export Views Excluding Overlays. This automatically sets the file to an 8-
bit size. Under Resolution (dpi), specify a number that when the Enter key is pressed results in
the file size being converted to ~300Kb (denoted in the lower right-hand corner of the Export
window). Several resolution values may need to be tested before finding the number that
converts the image to the appropriate file size. This value should be used when capturing all
subsequent PFGE gel images. Once the file size has been adjusted “Save Image.” These steps
convert and export TIFF files that are approximately the same size as those on the older
GelDoc systems (~300Kb) so they can be emailed and/or analyzed using BioNumerics.
5.3.10. Once the image has been successfully captured and saved, remove the gel and place in DI water until
analysis is completed. Use soft, lint-free towels (Kay-Dry or equivalent) to remove excess water
from surface of the transilluminator and clean using DI water and/or 70% isopropanol taking care not
to scratch the transilluminator platform.

5.4. GelDoc XR + (Image Lab Software – version 3.0 or higher).

5.4.1. After adequate staining and de-staining (if using ethidium bromide) of the agarose gel, a TIFF image of
the gel is required for analysis using the BioNumerics software. Handle the gel with gloves (powder-
free and not aloe-treated) at all times; remove or change gloves when using the computer keyboard or
mouse.
5.4.2. Turn on the GelDoc XR+ imager, the computer and the printer. On the Windows desktop, double click
on the Image Lab icon. Click on New Protocol or open protocol.
5.4.2.1. New Protocol: Step 1 – Gel imaging. Select application (nucleic acid gels, appropriate dye),
enter imaging area (22cm x 16.4cm for large or 15-well gels, 18cm x 13.4cm for small or 10-
well gels), select image exposure (intense or faint – may save two different protocols), select
Display Options (check highlight saturate pixels, image color gray).
Step 2 – lane and band detection – leave unchecked
Step 3 – analyze molecular weight – leave unchecked
Step 4 – Specify output, customize printing and reporting options if desired or leave unchecked.
Close and save protocol as .ptl file. Run protocol.
5.4.3. Open protocol or select recent protocol.
5.4.4. Open the drawer of the Gel Doc XR+ and carefully remove gel from container with gloved hands or
gel scoop. Drain excess liquid from gel and place it on the transilluminator platform.
5.4.5. Click position gel and hold in place using an empty frame. Close the drawer; the GelDoc XR+ will
automatically focus. If desired, use + and – buttons on computer screen to zoom the camera and fine
tune image size.
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 4 of 5
 STANDARD OPERATING PROCEDURE FOR IMAGE ACQUISITION AND CODE: PNL07
Effective Date:
PRODUCTION OF TIFF FILES FOR DATA ANALYSIS 07 26 2011

5.4.6. When satisfied with the image size, click Run Protocol. A window appears – click OK for filter 1.
5.4.7. Save the image as a .scn file. A name will be suggested corresponding to the date and time. The
filename should be changed as desired, i.e., CDC11001.scn.
5.4.8. Under Image tools on left panel, select Invert datayes.
5.4.9. In the File menu select Export to PulseNet. A .tiff file of the correct size with the same name as the
.scn file is saved.

6. FLOW CHART:

7. BIBLIOGRAPHY:

7.1. Weinberg, Sandy. GOOD LABORATORY PRACTICE REGULATIONS. Second edition. Marcel
Dekker, Inc. USA (1995).
7.2. Bio-Rad Laboratories, Inc., Quantity One User Guide, Gel Doc 2000 Documentation Systems
Hardware Instruction Manual, and Gel Doc EQ (ChemiDoc EQ, ChemiDoc XRS) Hardware
Instruction Manual.

8. AMENDMENTS:

8.1. 2011-07 Edited to clarify how far to zoom in on the gel during image capture. Added statements in each
section about using untreated gloves (no powder or aloe) when handling gels. Section 5.4 was added.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 5 of 5
 STANDARD OPERATING PROCEDURE FOR THE GENERATION OF RECORDS CODE: PNL08
Effective Date:
AND REPORTS 03 18 2005

1. PURPOSE: To describe the format and information needed for records and reports used in PulseNet.

2. SCOPE: All the records and reports that are generated from the testing and analysis of isolates by the PFGE
technique.

3. DEFINITIONS/TERMS:

3.1 PFGE: Pulsed-field Gel Electrophoresis

3.2 SOP: Standard Operating Procedure
3.3 DNA: Deoxyribonucleic acid

4. RESPONSIBILITIES:

4.1 Records:

4.1.1 Establish a sample receipt record (see Appendix PNL08-1).

4.1.2 Establish a record book or log with the following information:
4.1.2.1 Sample number
4.1.2.2 Date the samples were received
4.1.2.3 Date the plugs were made
4.1.2.4 Place where the plugs are kept
4.1.2.5 Electrophoresis equipment used
4.1.2.6 Gel number and lane number establishing the sample order in the photograph or image

4.1.3 Establish a work record for each gel (for an example see Appendix PNL08-2).
4.1.4 Establish a reagent control worksheet (for an example see Appendix PNL08-3).

4.2 Reports

4.2.1 The laboratory reports will follow the standard format used for the gel’s work record (see Appendix
PNL08-4).
4.2.2 The analysis reports will be done according to the standard format for reporting pattern numbers (see
Appendix PNL08-5).

5. PROCEDURE:

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:

9. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 7
STANDARD OPERATING PROCEDURE FOR THE GENERATION OF RECORDS CODE: PNL08
Effective Date:
AND REPORTS 03 18 2005

Appendix PNL08-1

EXAMPLE OF SAMPLE RECEIPT RECORD FOR PFGE ANALYSIS

CODE ORGANISM DATE OF ORIGIN DISTRIBUTION DATE OF

RECEIPT ANALYSIS

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 7
 STANDARD OPERATING PROCEDURE FOR THE GENERATION OF RECORDS CODE: PNL08
Effective Date:
AND REPORTS 03 18 2005

Appendix PNL08-2

EXAMPLE OF WORK RECORD

CONDITIONS

Number of samples: ______

Cell suspension concentration (range): Lowest _________ Highest ________

Total amount of proteinase K/cell in lysis buffer: Total: _________

Lysis incubation time ___________ and temperature __________

Location of standard in the gel: Lanes # ______________

RESTRICTION (Master Mix Preparation)

Total amount of buffer:_________ Total amount of water:_________ Total amount of enzyme: __________

Total volume: _______

AGAROSE GEL

Gel size: _____ 10-well or ______ 15-well gel.

0.5X TBE buffer:

10X TBE amount: ________ Reagent grade water: __________ Total volume: ____________

SKG agarose amount: ___________ Water: _________ 0.5X TBE amount: ____________

Electrophoresis run time: _______________

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 7
 STANDARD OPERATING PROCEDURE FOR THE GENERATION OF RECORDS CODE: PNL08
Effective Date:
AND REPORTS 03 18 2005

Appendix PNL08-3

EXAMPLE OF REAGENT CONTROL WORKSHEET

Date: ___________________

Laboratory: ____________________________________________

REAGENT LOT # SOURCE DATE MADE EXPIRATION DATE

TE Buffer

Sea Kem Gold agarose

SDS

Cell suspension buffer

Cell lysis buffer

Proteinase K

Reagent Grade Water

Enzyme

TBE (10x)

Ethidium bromide

_________________________ _________________________
Name Signature

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 7
 STANDARD OPERATING PROCEDURE FOR THE GENERATION OF RECORDS CODE: PNL08
Effective Date:
AND REPORTS 03 18 2005

Appendix PNL08-4

INTERNAL PFGE REPORT WORKSHEET

Samples Received From: Date PFGE Was Done:

Laboratory Submitting Image: Date Results Were Reported:
Date Isolates Were Received: TIFF File Number:

Conditions

Run Time hr

Initial Switch Time sec

Final Switch Time sec

Voltage Gradient 6 V/cm

Included Angle 120o

Ramping Linear

Initial milliamps

PFGE Analysis Performed by: Information Contact Person:

Reviewed by: Phone Number:
Lane CDC isolate # State isolate Organism Source Restriction PFGE Interpretation/Comments
# enzyme Pattern
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 7
 STANDARD OPERATION PROCEDURE FOR THE GENERATION OF RECORDS CODE: PNL08
Effective Date:
AND REPORTS 03 18 05

Appendix PNL08-5

Pulsed-Field Gel Electrophoresis Outbreak Report

Date:

To:

From:

Re: PFGE Molecular Subtyping Results Related to Outbreak

Isolates submitted to the CDC Outbreak Investigation Lab were analyzed by Pulsed-Field Gel Electrophoresis (PFGE) DNA fingerprinting using standardized PulseNet methods.

Isolates analyzed by PFGE subtyping were each given a unique isolate number when accessioned. Individual DNA fingerprint patterns were produced for each isolate using the
restriction enzyme XbaI (Primary enzyme) and BlnI (Secondary enzyme). The outbreak DNA fingerprint pattern from this outbreak has been assigned the pattern name [ ] by the CDC
PulseNet Data Administration Team.

Clinical Isolate Molecular Lab Date Date Source Organism PulseNet PulseNet Pattern PFGE Pattern
Number Number Collected Received Subtyped Pattern Name: Name: Interpretation
at CDC Primary Secondary
Enzyme Enzyme

General Interpretation Criteria:

Isolates that have been designated with the same PFGE pattern name as the outbreak pattern may be interpreted as indistinguishable from the outbreak strain. Isolates with different
PFGE fingerprint patterns have been designated as “different” in the PFGE Pattern Interpretation column. Specifically by this work, isolates that have indistinguishable DNA fingerprint
patterns are more likely to have originated from a common source; isolates that have different DNA fingerprint patterns are less likely to have originated from a common source.
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 6 of 7
 STANDARD OPERATION PROCEDURE FOR THE GENERATION OF RECORDS CODE: PNL08
Effective Date:
AND REPORTS 03 18 05

Pulsed-Field Gel Electrophoresis Outbreak Report

Additional Interpretation:

Disclaimer:
The results of PFGE fingerprinting analysis should always be used in conjunction with clinical, microbiologic, and epidemiologic
information. PFGE analysis is a population-based assay and should not be used for individual patient diagnostic purposes. PFGE is an
investigational tool and should be used for investigational purposes only. Epidemiological relatedness is considered to be the gold
standard.

Report completed by: ________________________ Date: ____________________

Reviewed by: ________________________ Date: ____________________

Reference: Swaminathan B, Barrett TJ, Hunter SB, Tauxe RV, the CDC PulseNet Task Force. PulseNet: The molecular subtyping network for
foodborne bacterial disease surveillance, United States. Emerg Infect Dis 2001;7:382-9

Website: www.cdc.gov/pulsenet

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 7 of 7
 STANDARD OPERATING PROCEDURE FOR THE USE, INSPECTION, CLEANING, CODE: PNL10
Effective Date:
MAINTENANCE, AND CALIBRATION OF EQUIPMENT 05 09 2005

1. PURPOSE: To establish guidelines for the use, inspection, cleaning, maintenance, and calibration
of equipment used for PulseNet-related activities.

2. SCOPE: All equipment used for the development and application of the PFGE technique, reagents,
solutions, and reports related to PFGE.

3. DEFINITIONS/TERMS:

3.1 PFGE: Pulsed-field Gel Electrophoresis

3.2 SOP: Standard Operating Procedure

4. RESPONSIBILITIES/PROCEDURE:
4.1 Equipment related to this SOP and requirements:

4.1.1 Spectrophotometers should have the wavelength confirmed with each use, using a
didymium filter, and calibrate using a standard solution with known absorbance on a
monthly basis (linearity check) or whenever new lots of controls, reagents, or
standards are obtained.
4.1.2 Colorimeters and Turbidity meters should be calibrated on a monthly basis according
to the instructions provided by the manufacturer.
4.1.3 Analytical balances should be calibrated with a set of class S weights certified by the
NIST or a qualified contractor on a quarterly basis.
4.1.4 Volumetric equipment, such as pipettes and autodiluters, should be checked yearly
for volume of delivery.
4.1.5 Upright refrigerators and freezers should have a calibrated thermometer immersed in
glycerol placed inside. Walk-in or stand-alone units may have adequate,
precalibrated digital thermometers. A recording thermometer may be used if already
installed in walk-in refrigerators. All temperatures should be checked daily.
4.1.6 Water bath temperatures should be checked and recorded daily with an adequate,
precalibrated thermometer.
4.1.7 Thermometers used in the laboratory should be checked annually against a certified
NIST thermometer over the full range of temperatures usually measured.

4.2 Develop for each item:

4.2.1 An inventory that lists all equipment, its location, model and serial number, age,
description, and the name of the person responsible for the item.
4.2.2 A definition of service tasks for each piece of equipment, procedures necessary for
upkeep and maintenance of equipment (calibration, proper functioning, cleaning,
etc.).
4.2.3 An interval frequency during which the defined procedures should be performed.
4.2.4 A personnel list of all individuals who are available for performing the upkeep and
maintenance.
4.2.5 In-service training of personnel in the use of special monitoring devices and the
performance of some of the more difficult service tasks should be provided, and
records of the names of those trained and the dates when the training occurred should
be maintained.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 1
STANDARD OPERATING PROCEDURE FOR THE USE, INSPECTION, CLEANING, CODE: PNL10
Effective Date:
MAINTENANCE, AND CALIBRATION OF EQUIPMENT 05 09 2005

4.2.6 A system so that the program may continue uninterrupted. A record sheet/book must
be established for each item of equipment, in which all entries are made.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 1
STANDARD OPERATING PROCEDURES FOR MAINTENANCE OF PULSED-FIELD CODE: PNL11
Effective Date:
GEL ELECTROPHORESIS SYSTEMS 02 05 2005

1. PURPOSE: To describe guidelines for maintenance of CHEF electrophoresis systems.

2. SCOPE: This procedure applies to equipment used with the PulseNet Standardized PFGE Protocols.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 PFGE: Pulsed-field Gel Electrophoresis
3.3 DNA: Deoxyribonucleic acid

4. RESPONSIBILITIES/PROCEDURES:

4.1 CHEF- Mapper, -DRIII, and Genepath systems

4.1.1 After each run:

4.1.1.1 Turn off cooling module, pump and main power switches of the instrument.
4.1.1.2 Install drain tube on right front port of chamber and drain buffer into sink or large
flask.
4.1.1.3 Prop up back of chamber with a pipet tip box or similar object to completely drain all
buffer from the chamber.

4.1.1.4 If another gel is to be run the same day:

a. Remove drain tube.

b. Add 2 liters of appropriate buffer (0.5X TBE) to the chamber.
c. Wait at least 30 minutes after the last run (4.1.1.1) to turn on the pump and
chiller. If chiller is turned on and the pump is not, ice may form in the heat
exchanger, which can block the flow of buffer and prevent it from reaching the
desired temperature (14ΕC). If this occurs, turn off chiller and pump, and allow
the unit to remain at room temperature for at least 30 minutes so any ice that
may have formed in the heat exchanger can thaw.

4.1.1.5 If the chamber will not be reused on the same day:

a. Rinse the gel chamber with 2 liters of deionized (Type 1) water.

b. Drain by propping back of chamber with a pipet tip box or similar object as
described in 4.1.1.3.
c. Wipe the inside lid and sides of chamber with a damp towel to remove any
residual buffer. Do not touch the electrodes. Remove any remaining small
pieces of agarose in chamber with a soft paper towel (Kaydry, Kimwipe, or
equivalent towel).

4.1.2 Weekly

4.1.2.1 Once a week or if the PFGE unit is not to be used for several days, the gel chamber
and cooling module tubing should be flushed with water.

a. Add 2 liters of deionized (Type 1) water to chamber.

b. Turn on the pump to circulate it through the tubing and electrophoresis chamber.
Do not turn on the chiller.
c. Let the water circulate for 10-15 minutes, then turn off the pump.
d. Drain as much of the water out of the chamber as possible.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 5
STANDARD OPERATING PROCEDURES FOR MAINTENANCE OF PULSED-FIELD CODE: PNL11
Effective Date:
GEL ELECTROPHORESIS SYSTEMS 02 05 2005

e. Disconnect tubing from the left front port, turn pump on briefly (30 seconds) to
force any residual water from tubing into the gel chamber.
f. Drain chamber as in Step 4.1.1.3.
g. Wipe the inside lid and sides of chamber with a damp towel to remove any
residual buffer. Do not touch the electrodes.
h. Re-connect tubing to left port of the chamber. Note: After this step is completed,
the chamber will require 2.2 liters of buffer to fill.
i. Check that the gel chamber is level before adding buffer.

4.1.3 As necessary

4.1.3.1 If the chamber has not been used recently or if your gel is blank even after Lambda or
other standards are run, the lines may be contaminated. Bacterial or fungal growth in
the tubing will degrade the DNA and a blank gel will result.

a. Flush the chamber as described in 4.1.2 with 2 liters of bleach solution (5% -
10% in water) instead of deionized (Type 1) water for 15-30 minutes.
b. Drain bleach solution from chamber, add 2 liters of deionized (Type 1) water,
and circulate 15-30 minutes.
c. Drain water and repeat the water wash at least two more times to be sure that all
of the bleach solution is removed from the tubing.

4.1.3.2 If the problem persists (cleaning with bleach solution does not remove all of the
contamination from the tubing), replace all the lines with new tubing.

4.1.4 Replacement of electrodes (if broken or damaged):

4.1.4.1 Unplug chamber from wall socket or remove electrical connector(s) from chamber.
4.1.4.2 Disconnect tubing at back and left front port of gel chamber.
4.1.4.3 Turn gel chamber upside down and remove all the screws.
4.1.4.4 Lift off the base plate.
4.1.4.5 Remove the hexagonal nut on the wire, remove the nut on the electrode to be
replaced, and push down firmly on the post to remove the old electrode.
4.1.4.6 Turn the gel chamber over, insert the new electrode, pack with self-leveling silicone
sealant (RTV-type sealant, which is available at most hardware stores).
4.1.4.7 Replace the nut, wire, and base plate.
4.1.4.8 If one of the pins to the serial cable bends, use tweezers to carefully straighten it.

4.1.5 Replacement of a CHEF blown fuse (available at electronic or hardware stores).

4.1.5.1 Make sure the cord to the power module is unplugged when replacing the fuse.
4.1.5.2 If the voltage to the electrodes exceeds 300 V, the 0.5 ampere FB (Fast Blow) fuse
may blow. The high-voltage indicator light will go on and the power module will go
off. Replace the fuse by unscrewing the cartridge at the front of the power module
and change the blown fuse with a 0.5 ampere FB (Fast Blow) fuse.
4.1.5.3 If there is a power surge, the SB (Slow Blow) fuse may blow. The AC light on the
power module will go off. The fuses are at the rear of the power module, mounted
inside the power entry module. Replace the fuse with 3.0 A SB fuse if your local
voltage is 120 V or 100 V, or with a 1.5 A SB fuse if your local voltage is 220 V or
240 V.

4.1.6 Replacement of a GenePath blown fuse (available at electronic or hardware stores).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 5
STANDARD OPERATING PROCEDURES FOR MAINTENANCE OF PULSED-FIELD CODE: PNL11
Effective Date:
GEL ELECTROPHORESIS SYSTEMS 02 05 2005

4.1.6.1 Make sure the cord to the power module (Gene Path system) is unplugged when
replacing the fuse.
4.1.6.2 If the DC current entering the gel chamber exceeds 500 mA, then the 0.5 ampere FB
(Fast Blow) fuse will blow and an error code F2 will be displayed. Replace fuse by
unscrewing the cartridge at the front of the GenePath system and change the fuse with
a 0.5 ampere FB fuse.
4.1.6.3 A power surge will cause the SB (Slow Blow) line fuse to blow. The LED lights on
the GenePath system will go off. This fuse is located at the back of the drive module.
Replace the fuse with a 3.15 A SB fuse if the line voltage is 100 or 120 V, or a 1.6 A
SB fuse if the line voltage is 220 or 240 V.

4.2 Cooling Module

4.2.1 Installation

4.2.1.1 See manual included with the instrument for installation instructions.

4.2.2 Operation: After the Cooling Module has been connected, the following steps should be
followed for optimal operation:

4.2.2.1 Switch on the recirculating pump, and adjust flow rate to 1.0 liter/minute (setting of
~70-80).
4.2.2.2 Switch on the Cooling Module (main power switch is on the front panel of the unit).
The temperature display will read “14.0” (or the temperature set in the previous run)
after 10 beeps, indicating that the unit is on and the red light above the “SET TEMP”
button will illuminate.
4.2.2.3 Enter the desired run temperature (14°C is recommended for PFGE runs) by pressing
the “LOWER” or “RAISE” arrows until the desired number is reached.
4.2.2.4 After 100 seconds have elapsed, the compressor will engage (if the set temperature is
below ambient temperature), and the red light above “COOLING” will illuminate.
This 100-second delay allows pressure equalization in the system to avoid mechanical
damage.
4.2.2.5 Press the “ACTUAL TEMP” button (the red light above the button will illuminate) to
read the current temperature monitored at the internal temperature probe (located in
the “OUT FLOW” port) or at the cell's internal temperature probe (if connected).
4.2.2.6 After the Cooling Module has cooled the buffer to the desired set temperature, the
“COOLING” light will occasionally go on and off accompanied by a click. This is an
indication of the refrigerant bypass valve cycling on and off to maintain average
buffer temperature within 1° C of set temperature. [Greater temperature fluctuations
may occur with longer tubing length, low buffer flow rates, and/or high amounts of
input power and high ambient temperatures. However, the built-in adaptive algorithm
will compensate for these conditions so that the average temperature is typically
within 1°C.]

NOTE: Please allow sufficient room for ventilation at both the front and rear of the unit. The
cooling fan is located in the rear.

4.2.2.7 Bio-Rad recommends that the buffer in the system be pre-cooled to the desired run
temperature. The Cooling Module can quickly pre-cool the system, at an approximate
rate of 0.75° C/minute in the absence of input power to the recommended operating
temperature of 14° C. In other words, the cooling module can pre-cool buffer at
ambient temperature to 14° C in approximately 10-15 minutes and is designed to
maintain set temperature during the run. If the buffer is not pre-cooled, the presence
VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 5
STANDARD OPERATING PROCEDURES FOR MAINTENANCE OF PULSED-FIELD CODE: PNL11
Effective Date:
GEL ELECTROPHORESIS SYSTEMS 02 05 2005

of input power (from the electrophoresis power supply) will greatly increase the time
required to bring the system to the set temperature.

NOTE: The 0.75° C/minute pre-cooling rate decreases for set temperatures lower than 14° C.

4.3. Maintenance

4.3.1 When the Cooling Module will not be used for an extended time, rinse the lines in the
heat exchanger thoroughly with distilled water to remove all traces of electrophoresis
buffer. Drain the residual water from the heat exchanger, and dry it with laboratory
air.

4.3.2 Main Switch Fuse Check and Replacement

a. Turn the unit around so that the back of the unit is facing forward, and locate the
black plastic assembly containing the power cord receptacle on the lower left
corner.
b. Turn the unit off and remove the main power cord from the receptacle (grasp the
female plug, not the cord, and pull gently).
c. Turn the unit off and remove the main power cord from the receptacle (grasp the
female plug, not the cord, and pull gently).
d. Using the screwdriver, gently pull the fuse holders forward (out). Remove the
fuse from the holder and examine. If the fuse is obviously burned, replace it with
a 6.3 Amp, 250 V SB (Slow Blow) fuse (100/120 V) or 3.0 Amp, 250 V SB fuse
(220/240 V). Otherwise, check the fuse with a volt-ohmmeter.
e. Place the fuse holders back into the slots so that the arrows on the fuse holders
point in the same direction as the two arrows on the inside of the front cover.
Press the fuse holders firmly into place; close the front cover by pressing firmly
at the top comers.
f. Replace the power cord. The unit is now ready for operation. If the unit still
does not turn on, or the fuse burns again, please contact Bio-Rad instrument
service.

4.4 Variable Speed Pump

4.4.1 The variable speed pump is powered from the CHEF- DRII Drive module, CHEF- DRIII
power module, CHEF-Mapper power module, or Gene-Path power module.

4.4.1.1 Plug the pump into the socket marked “Pump” at the front or back of the module.

4.4.1.2 Turn the pump on using the switch marked “Pump Switch.” Adjust the flow rate by
turning the dial on the variable speed pump. The setting of 100 on the dial is 100% of
the pump’s maximum flow rate, though the flow is modified by varying line voltage.
A setting of 60 -70 on the dial is recommended for most pulsed-field gel
electrophoresis runs with the CHEF system. This corresponds to a flow rate of
about 1 liter/minute.
4.4.1.3 If the pump does not start, check the 0.5 amp (SB) fuse on the back of the pump,
located under the round gray lid. NOTE: The variable speed pump is ground isolated
for safety. Disconnect electric power to pump before changing the fuse.

5. FLOW CHART:

6. BIBLIOGRAPHY:
VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 5
STANDARD OPERATING PROCEDURES FOR MAINTENANCE OF PULSED-FIELD CODE: PNL11
Effective Date:
GEL ELECTROPHORESIS SYSTEMS 02 05 2005

Bio-Rad Laboratories. CHEF Mapper XA Pulsed-field Electrophoresis Systems, Instruction Manual and Application
Guide. 1995; pp. 65.

Bio-Rad Laboratories. CHEF-DR III Pulsed-field Electrophoresis Systems, Instruction Manual and Application
Guide. 1992; pp. 27.

Bio-Rad Laboratories. GenePath System, Instruction Manual. pp. 10-11.

Bio-Rad Laboratories. CHEF Variable Speed Pump Manual.

Bio-Rad Laboratories. Cooling Module Instruction Manual.

7. CONTACTS:

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 5
 STANDARD OPERATING PROCEDURE FOR MAINTENANCE OF DADE CODE:PNL12
MICROSCAN TURBIDITY METER Effective Date:
02 14 2005

1. PURPOSE: To describe the guidelines for the use and maintenance of the Dade MicroScan Turbidity Meter.

2. SCOPE: This procedure applies to equipment used with the PulseNet Standardized PFGE Protocols.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 PFGE: Pulsed-field Gel Electrophoresis

4. PROCEDURES:

4.1 Operational Precautions and Limitations

4.1.1 For in vitro diagnostic use only.

4.1.2 Read entire manual prior to operation.
4.1.3 Avoid spilling liquids into the instrument as this may damage sensitive components.
4.1.4 Use identical tubes for blanks and samples that are visually clean, clear and free from
scratches.
4.1.5 The rechargeable NiCad batteries should last for at least 500 readings between recharging.
When the battery voltage has declined to the point at which a reading error could result, a
battery check circuit will extinguish the display. This is your indication that the batteries
require recharging. Batteries can be recharged overnight, or the instrument can be left plugged
in indefinitely without damage.

4.2 Maintenance

NOTE: Laboratorian should never attempt repair procedures on equipment such as turbidity
meters, spectrophotometers, and similar electronic devices. Equipment should be sent to
machine shop or back to the manufacturer for any major repair.

4.3 Operation

4.3.1 Turbidity Determination

4.3.1.1 Prepare the desired base medium in which suspensions will be made.
4.3.1.2 Dispense 2.5 ml and at least 3 ml of the medium into two clean Falcon 2054 or Falcon
2057 tubes, respectively. These will be used to confirm that the reading on the digital
output is 0.00 ± 0.01.before measuring the turbidity of the test samples. One of these
tubes will be referred to as the “blank.”
4.3.1.3 Prepare the desired bacterial test suspensions in the same tube size and base medium
as the blank. (Refer to PulseNet Standardized PFGE Protocol.)
4.3.1.4 Insert one of the blanks prepared in Step 4.3.1.2 into the “BLANK” or “1” position of
the turbidity meter. Insert the test suspension into the “SAMPLE” or “2” position.
4.3.1.5 The display indicates the difference in absorbance between the blank and sample
tubes. The acceptable range equivalent to a 0.5 McFarland Standard is 0.05 – 0.12.
4.3.1.6 Measure the turbidity of the test suspensions at least three times to confirm that the
reading is stable.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 2
 STANDARD OPERATING PROCEDURE FOR MAINTENANCE OF DADE CODE:PNL12
MICROSCAN TURBIDITY METER Effective Date:
02 14 2005

5. FLOW CHART:

6. BIBLIOGRAPHY:

Dade MicroScan Turbidity Meter instruction pamphlet, Dade International Inc., 1996.

7. CONTACTS:

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 2
 STANDARD OPERATING PROCEDURE FOR WATERBATH MAINTENANCE AND CODE:PNL13
CLEANING Effective Date:
02 14 2005

1. PURPOSE: To describe guidelines for the maintenance and cleaning of laboratory water baths.

2. SCOPE: All water baths involved with PulseNet-related activities must follow a documented schedule for
maintenance and cleaning.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 PFGE: Pulsed-field Gel Electrophoresis

4. RESPONSIBILITIES/PROCEDURES:

4.1 Check and record operating temperature of all water baths used in the PulseNet procedures each day
prior to use (Appendix PNL13-1).

4.1.1 Accurate and reliably maintained temperature is essential for consistent results.
4.1.2 Temperature limit: desired temperature ±1.0oC.
4.1.3 If temperature is out of range, adjust control. (See instructions specific to each water bath.)

4.1.3.1 After one hour, recheck temperature and record.

4.1.3.2 If still out of range, notify party responsible for service and repair; DO NOT USE.
4.1.3.3 Use alternative water bath until repairs have been completed and temperature is
within the accepted range.

4.2 Check water baths each day prior to use for leaks and inspect for bacterial growth/contamination.

4.2.1 If necessary, add enough distilled water to ensure there are adequate water levels to maintain
the temperature(s) required for the PulseNet PFGE procedures.

4.3 Monthly drain and thoroughly clean unit per manufacturer’s recommendations.

4.3.1 Turn off and unplug the water bath.

4.3.2 Use the drain (if present) or a siphon assembly for this purpose. After most of the water is
drained, remove any remaining water with a sponge or soft towel.
4.3.3 Wash tank and shaker components (if present) with a solution of mild soapy water. Use a soft
cloth or sponge. Make sure to wear protective gloves when cleaning water baths. Rinse all
components thoroughly and dry completely. DO NOT USE chlorine bleach, chlorine-based
cleanser, abrasives, ammonia, steel wool or scouring pads with metallic content - these will
damage the water bath. Materials effective in disinfecting include glutaraldehyde or 70%
alcohol.
4.3.4 Clean the low water sensor (when present) every two weeks. Scale build-up may be removed
with a toothbrush while it is immersed in water.
4.3.5 Refill the tank with DISTILLED WATER only. Do not use tap water; it will cause mineral
deposits and possible corrosion. A non-chlorine-based fungicide/bactericide may be added to
the tank to inhibit fungal or bacterial contamination of the bath water.

5. FLOW CHART:

6. BIBLIOGRAPHY:

Evaluation, Verification and Maintenance Manual. College of American Pathologists. Lab-Line Instruments,
Inc. Lab-Line Orbital Water Bath Shakers Operation Manual. 1998.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 2
 STANDARD OPERATING PROCEDURE FOR WATERBATH MAINTENANCE AND CODE:PNL13
CLEANING Effective Date:
02 14 2005

7. CONTACTS:

8. AMENDMENTS:

Appendix PNL13-1

Waterbath:

Location:

DATE TEMPERATURE INITIALS

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 2
STANDARD OPERATING PROCEDURE FOR THE USE, STORAGE, AND DISPOSAL CODE:PNL14
Effective Date:
OF CHEMICALS AND REAGENTS 03 18 2005

1. PURPOSE: To describe the safe use, storage and disposal of chemicals and reagents in laboratories
performing pulsed-field gel electrophoresis.

2. SCOPE: For all personnel and laboratories conducting PulseNet-related activities.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 PFGE: Pulsed-field Gel Electrophoresis
3.3 PPE: Personal Protective Equipment (goggles, gloves, lab coat, etc.)
3.4 MSDS: Material Safety Data Sheets
3.5 OHS: Office of Health and Safety

4. RESPONSIBILITIES:

4.1 Use of chemicals and reagents

4.1.1 Know what chemicals and reagents are being used in any procedure.

4.1.1.1 Read chemical labels and MSDS if you are unfamiliar with any chemical or
reagent.

a. All MSDS related to chemicals and reagents in the laboratory should be

readily accessible to laboratory personnel.

4.1.2 Have proper safety equipment available in the laboratory.

4.1.2.1 Obtain and use all appropriate PPE.

4.1.2.2 Use the appropriate ventilation device when working with chemicals
(chemical fume hood) or infectious material (biosafety cabinet).

a. Do not use a biosafety cabinet if the work requires a chemical fume hood
and vice versa.

4.1.3 Use the smallest quantity of chemicals or reagents possible.

4.1.4 Know what to do in case of an accident.

4.1.4.1 Know the location of eye washes, showers, fire extinguishers, and exits.
4.1.4.2 Know the location and use of chemical spill kits.
4.1.4.3 Have the contact information for the OHS Environmental Program or
appropriate safety office posted near the phone together with all emergency
numbers.

4.2 Storage of chemicals and reagents

4.2.1 All chemicals are to be stored properly according to recognized compatibilities.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 3
STANDARD OPERATING PROCEDURE FOR THE USE, STORAGE, AND DISPOSAL CODE:PNL14
Effective Date:
OF CHEMICALS AND REAGENTS 03 18 2005

4.2.1.1 Chemicals posing special hazards or risks shall be limited to the minimum
quantities required to meet short-term needs and stored under appropriate safe
conditions (i.e., chemical safety cabinets).
4.2.1.2 Chemicals are not to be stored on the floor or in chemical fume hoods.
4.2.1.3 Compressed gas cylinders shall be secured at all times.

4.2.2 A chemical inventory of all hazardous chemicals will be maintained and posted on the
door to each laboratory.
4.2.3 All chemicals and reagents shall be labeled with the date they were received into the
laboratory.
4.2.4 Expired chemicals, reagents, and chemical waste will be disposed via the hazardous
chemical waste disposal program
(see http://intranet.cdc.gov/ncid/site/safety/chemhygiene/disposal.htm) or appropriate
safety guidelines.
4.2.5 Each laboratory must have a written chemical hygiene plan specific for that laboratory
(see http://intranet.cdc.gov/ncid/site/safety/chemhygiene/chp.htm) or appropriate
safety guidelines.

4.3 Disposal of chemicals and reagents

4.3.1 Know the hazardous chemical status of your chemicals and reagents.
4.3.2 Only “certified chemical waste managers” may dispose of hazardous chemical waste.
Record the name of the “certified chemical waste manager” for your laboratory and
refer any questions about disposal of chemicals to him or her.
4.3.3 Keep all waste containers or expired chemicals closed tightly.

4.4 Have your “certified chemical waste manager” complete the appropriate form(s). Indicate
building and room where waste is located on a “Hazardous Chemical Waste Disposal Label”
(Form CDC 0.886, Rev. 10/94).

4.4.1 Send top copy to OHS Environmental Program office, Mailstop F-05, when ready for
pickup.
4.4.2 Peel backing off label and attach to waste container. Do not cover up the original
container label. Mark “XX” through label if original container is not being used.
4.4.3 For empty chemical containers, remove CDC Chemical Tracking System barcode
sticker and affix to the “Chemical Disposition Form,” or send “form” to OHS
Environmental Program office (MS-F-05) to request a pickup.
4.4.4 For more information on hazardous or chemical waste disposal contact the
Environmental Program, Office of Health and Safety, at 404-639-1464.

4.5 DO NOT POUR HAZARDOUS CHEMICALS DOWN THE DRAIN!

5. PROCEDURE:

6. FLOW CHART:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 3
STANDARD OPERATING PROCEDURE FOR THE USE, STORAGE, AND DISPOSAL CODE:PNL14
Effective Date:
OF CHEMICALS AND REAGENTS 03 18 2005

7. BIBLIOGRAPHY:

Laboratory Survival Skills: A Primer on Responsibilities, Safety Practices, and Emergency Response
for CDC Employees.

Prudent Practices in the Laboratory: Handling and Disposal of Chemicals, Nat. Academy Press
(1995).

CDC Chemical Waste Handling and Disposal Guide

CDC Chemical Hygiene Plan

8. CONTACTS:

8.1 CDC OHS Environmental Program (404) 639-1464

9. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 3
 STANDARD OPERATING PROCEDURE FOR THE STORAGE AND USE OF CODE: PNL15
Effective Date:
EXPIRED REAGENTS 02 14 2005

1. PURPOSE: To describe under what circumstances expired reagents may be stored and used.

2. SCOPE: Expired reagents are not to be used for PulseNet-related activities except as described
below.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 CDC: Centers for Disease Control and Prevention
3.3 PFGE: Pulsed-field Gel Electrophoresis

4. RESPONSIBILITIES/PROCEDURES:

4.1 Expired reagents are not to be used for any PulseNet-related PFGE gels if the results are
intended, or might be expected, to be included in the PulseNet national database(s).

4.1.1 Discard commercially prepared reagents on or before the expiration date or if

precipitation, discoloration, or cloudiness is observed.
4.1.2 Discard laboratory-prepared reagents after six to nine months or if precipitation,
discoloration, or cloudiness is observed.

4.2 Expired reagents may be retained for training, troubleshooting, and research purposes
provided that they are labeled clearly with: “For training, troubleshooting, or research
ONLY.”

4.3 Expired reagents must be discarded and disposed of according to CDC’s and each
laboratory’s protocols (see PNL14).

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 1
STANDARD OPERATING PROCEDURE FOR EVALUATION AND CORRECTION OF CODE:PNL16
FAILURE OF ANY COMPONENT OF STANDARDIZED PFGE PROTOCOL Effective Date:
02 14 2005

1. PURPOSE: This procedure must be followed if a failure is detected in any component of the standardized
PFGE protocol (bad reagents, poor standards, equipment failure, inadequately trained lab personnel) resulting
in gels unacceptable for analysis and inclusion in the PulseNet National Database(s).

2. SCOPE: To be used by all laboratories involved in PulseNet-related activities.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 PFGE: Pulsed-field Gel Electrophoresis
3.3 PFGE inbox: An email account that is maintained and checked by all database managers at CDC. The
address is: [email protected].
3.4 CDC: Centers for Disease Control and Prevention
3.5 DNA: Deoxyribonucleic acid

4. RESPONSIBILITIES/PROCEDURES:

4.1 Failure of Reagents

4.1.1 Identify what reagent(s) caused the problem. Visual inspection of the TIFF image of the
problem gel by an experienced individual may lead to clues that may help narrow down
potential problems.

4.1.2 Determine the nature of the problem.

4.1.2.1 Contaminated reagent

4.1.2.2 Expired reagent
4.1.2.3 Incorrect concentration, pH, etc. of one or more reagents.

4.1.3 If reagent is commercial product, open new lot/bottle and test by running protocol with a
culture with a known PFGE pattern (e.g., H9812 standard strain).

4.1.4 If reagent is prepared “in-house,” prepare new lot of reagent following established procedures
and test by running protocol with a culture with a known PFGE pattern (e.g., H9812 standard
strain).

4.2 Failure of Standards

Note: Gels with poor standard strains cannot be normalized for comparison to the national
databases. In some instances the gels can be visually compared; however, all gels with poor or
missing standard strains must be rerun prior to inclusion in the national databases.

4.2.1 Insufficient or too much DNA in plug

4.2.1.1 Discard and remake plug

4.2.1.2 Check equipment used to measure the cell concentration (turbidity meter, etc.) to
ensure that it is operating properly.

4.2.2 Incomplete restriction

4.2.2.1 Repeat TE wash 2X, and then repeat restriction step on new slice of the same plug.
Include at least one slice from a previously tested plug that gave a satisfactory
pattern on another gel (+ control).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 4
STANDARD OPERATING PROCEDURE FOR EVALUATION AND CORRECTION OF CODE:PNL16
FAILURE OF ANY COMPONENT OF STANDARDIZED PFGE PROTOCOL Effective Date:
02 14 2005

4.2.2.2 If problem persists, restrict DNA with a new vial of enzyme. Use different lot
number when possible.
4.2.2.3 Confirm calculations for making enzyme/buffer mixture are correct.

4.2.3 One or more lanes with standard strain is distorted, smeared, etc.

4.2.3.1 Repeat gel

4.2.4 Inadequate number of standards run on gel.

4.2.4.1 Repeat gel with standard strain in the end lanes and after every three or four test
strains (e.g., lanes 1, 5, 10 on 10-well gel; lanes 1, 5, 10, 15 on 15-well gel).

Note: Gels with an insufficient number or placement of standards may become distorted when
normalized and be inadequate for analysis and comparison to the national databases.

4.3 Equipment failures may occur even with proper maintenance and service.

Note: All equipment failures, regardless of the severity, must be documented for review.

4.3.1 Temperature fluctuation in the electrophoresis chamber due to problems with the cooling
module, pump, length and condition of tubing, bubbles in tubing, extreme changes in ambient
temperature or voltage fluctuation with the power supply.

4.3.1.1 Temperature fluctuations of >±3ºC may negatively impact the resolution and quality
of the bands.
4.3.1.2 Each lab must monitor and document the temperature for each chamber used for
PFGE for PulseNet to make sure that it operates within the parameters recommended
in the standardized protocols.

4.3.2 Inadequate buffer flow due to debris or blockage in the chamber

4.3.2.1 Poor buffer flow often results in poor resolution and uneven migration of the DNA
fragments through the gel.
4.3.2.2 The lines and left buffer port must be checked and cleaned periodically to ensure that
the buffer can flow evenly.
4.3.2.3 The use of an agarose gel trap within the chamber can help to filter out agarose
debris preventing blockage.
4.3.2.4 Always check the flow settings on the pump to make sure that it meets the rate
indicated in the standardized protocol (~1L/min.).

4.3.3 Broken or failed electrodes in the chamber can cause uneven distribution of current and will
distort the band movement through the gel.

4.3.3.1 Examine electrodes periodically.

4.3.3.2 Replace broken or failed electrodes as needed (see SOP PNL11).

4.3.4 Power outage or blown fuses

4.3.4.1 The most recent models of the Chef Mapper are designed to restart the program
where it shuts down, but if the battery back-up is defective this may not occur.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 4
STANDARD OPERATING PROCEDURE FOR EVALUATION AND CORRECTION OF CODE:PNL16
FAILURE OF ANY COMPONENT OF STANDARDIZED PFGE PROTOCOL Effective Date:
02 14 2005

4.3.4.2 The Chef Mapper is equipped with both Fast Blow (FB) and Slow Blow (SB) fuses,
and the program may not restart where it shut down if the fuse blows and has to be
replaced.

4.4 Inadequately trained lab personnel

4.4.1 Laboratory personnel who have not been certified in the PulseNet standardized protocol(s) for
PFGE shall not submit TIFF or bundle files on-line to the PulseNet national database(s).
Laboratories that are not certified must send their TIFF images to the PFGE inbox with the
appropriate organism in the subject line of the email.

4.4.1.1 Personnel may be trained in several ways:

a. Attend a CDC-sponsored PFGE training workshop

b. Receive training through an approved PulseNet Area Laboratory
c. Receive one-on-one bench training at CDC, or in the individual’s lab, by
personnel from CDC or approved PulseNet Area Laboratory.

4.4.1.2 Personnel must complete their certification process prior to submitting TIFF or
bundle files to the PulseNet national database(s) unless authorized to do so by a
member of the PulseNet National Database Administration Team.
4.4.1.3 Personnel who continue to produce unsatisfactory results subsequent to an initial
training SHALL be retrained.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

7.1 CDC PulseNet Database Administration Team

(404) 639-4558
[email protected]

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 4
STANDARD OPERATING PROCEDURE FOR EVALUATION AND CORRECTION OF CODE:PNL16
FAILURE OF ANY COMPONENT OF STANDARDIZED PFGE PROTOCOL Effective Date:
02 14 2005

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 4
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE LABORATORY CODE: PNL17
Effective Date:
PROTOCOL TRAINING 5 09 2005

1. PURPOSE: To describe the procedure for training of PulseNet personnel in PulseNet laboratory
PFGE protocols.

2. SCOPE: This procedure applies to all PulseNet participants and hosts of PulseNet PFGE laboratory
protocol training courses.

3. DEFINITIONS/TERMS:

3.1 Host lab: Term used to describe a PulseNet laboratory that has been approved by CDC to host a
training course
3.2 Training personnel: Term used to describe PulseNet participant(s) who have been approved by CDC
to train other PulseNet participants
3.3 APHL: Association of Public Health Laboratories
3.4 SOP: Standard Operating Procedure
3.5 CDC: Centers for Disease Control and Prevention
3.6 PFGE: Pulsed-Field Gel Electrophoresis
3.7 PPE: Personal Protective Equipment

4. RESPONSIBILITIES:

4.1 All PulseNet personnel are required to read the PulseNet QA/QC manual and all PulseNet SOPs.
4.2 At least one PulseNet participant from each participating PulseNet laboratory is required to
attend annual PulseNet update meetings and regional meetings when they occur.
4.3 All PulseNet personnel performing PFGE for PulseNet pathogens and hosting PFGE training
must have knowledge of aseptic techniques, pure culture isolation, principles of PFGE, and basic
laboratory safety.
4.4 Host lab(s), APHL, and/or CDC will determine training needs of PulseNet participants.
4.5 All PulseNet personnel must be trained by a host lab or other approved training personnel.
4.5.1 PulseNet laboratory training covers the principles of PFGE, a detailed review of the
PulseNet standardized PFGE protocol, and the use of gel image acquisition hardware and
software.
4.6 Host labs and training personnel should adequately prepare participants in the PulseNet PFGE
protocols. When training is finished, the trainee(s) should be able to submit PulseNet gels for gel
certification (see SOP PNQ02).
4.7 Host labs and training personnel must be gel-certified.
4.8 Host labs must put together training materials for the trainees.
4.9 PulseNet participants will then be evaluated through certification (PNQ02) and proficiency
testing (PNQ04).

5. PROCEDURE:

5.1 Adequate communication with APHL and CDC must be maintained in order to determine
training needs and assure proper attendance at required meetings.
5.2 Host labs and/or training personnel should work with trainees and/or APHL to determine a
feasible time and location for the course.
5.3 The following is a recommended procedure for hosting a PulseNet laboratory training course:
5.3.1 The host lab should organize an agenda committee to create an agenda and a timeline of
organizational duties, and to obtain necessary laboratory training materials.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 4
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE LABORATORY CODE: PNL17
Effective Date:
PROTOCOL TRAINING 5 09 2005

5.3.2 It is recommended that each participant be provided, but not limited to, the following
items:
5.3.2.1 Participant and trainer contact information
5.3.2.2 PulseNet protocols for E. coli, Campylobacter, Salmonella, Listeria monocytogenes,
and Shigella
5.3.2.3 Laboratory reagents
5.3.2.4 Laboratory supplies for PFGE
5.3.2.5 Pathogenic strains to be tested
5.3.2.6 Proper laboratory PPE
5.3.2.7 Course evaluations
5.3.2.8 Course certificates to indicate successful completion of training
5.3.3 The committee may also be responsible for making lodging, transportation, and meal
arrangements for course participants.
5.3.4 For information regarding correct laboratory chemical handling, refer to PNL14 (“SOP
for the Use, Storage, and Disposal of Chemicals and Reagents”).
5.3.5 Refer to PNL01 (“SOP for Lab Equipment and Supplies”) and PNL02 (“SOP for Lab
Formulas and Reagents”) for laboratory equipment and safety information.
5.3.6 It is recommended that training be carried out using E. coli or Salmonella.
5.3.7 Refer to Appendix PNL17-01 for a recommended sample laboratory training agenda.
5.3.8 Assign training responsibilities to trainers.
5.3.9 It is recommended that there be one trainer per five participants.
5.3.10 All reagents and strains being used during the training course should be tested before the
training course.
5.3.11 At the completion of training, participants should fill out evaluations and be awarded
certificates of completion.
5.3.12 Summarize evaluations and supply trainers and members of the agenda committee a
summary of the conference evaluations.

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:

8.1 Training support at CDC: PulseNet Database Administration Team

(404) 639-4558
[email protected]

9. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 4
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE LABORATORY CODE: PNL17
Effective Date:
PROTOCOL TRAINING 5 09 2005

Appendix PNL17-01

SAMPLE Agenda for Standardized Molecular Subtyping of Foodborne Bacterial Pathogens by

Pulsed-field Gel Electrophoresis (PFGE)

Day 1

Welcome, Introductions, and Remarks

General Information/Workshop Objectives

Overview of PulseNet

Epidemiology of Foodborne Diseases? or Molecular Epidemiology Using PFGE Subtyping Data? Utility
of PFGE data?

Overview of PFGE Manual and Laboratory Procedures

Laboratory Safety Information

Laboratory Module IA, IB

Prepare Salmonella and/or E. coli O157:H7 agarose PFGE plugs
Lyse cells in agarose plugs

The Standardized PFGE Protocol(s) for Foodborne Enteric Pathogens

Use of Additional Restriction Enzymes for PFGE in Outbreak Situations

Laboratory Module IC
Begin washing agarose plugs with water to remove lysis reagent.
a. Wash plugs two times with 15 ml sterile Type 1 water for 10 min. at 50ºC.

Begin washing agarose plugs with TE to remove lysis reagent

a. Wash plugs three to four times with 15 ml sterile TE for 15 min. at 50ºC.
b. Discussion/demonstration of alternate ways to wash PFGE plugs.

Day 2

Laboratory Module IIA

Restriction digestion of DNA in agarose plugs

Optional Bio-Rad Product Information

Questions and Answers for Bio-Rad
Local Bio-Rad Representative

Laboratory Module IIB

Load restricted plug slices on comb and cast PFGE gel
Demonstrate PFGE equipment
VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 4
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE LABORATORY CODE: PNL17
Effective Date:
PROTOCOL TRAINING 5 09 2005

Laboratory Module IIC, IID

Begin electrophoresis of PFGE gels

Break/Open Discussion

Day 3

Laboratory Module III

Begin staining the agarose gels with Ethidium Bromide
Demonstrate care of electrophoresis equipment after run is over

Begin de-staining gels

Troubleshooting gels when there are no bands (include thiourea and treatment of electrophoresis
chamber with bleach)

Question/answer session about lab portion of PFGE

Demonstrate of Gel Doc 2000

Capture and save images of gels using the Gel Doc 2000

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 4
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
5 25 2007

1. PURPOSE: To describe the standardized protocol for molecular subtyping of Yersinia pestis by
Pulsed-field Gel Electrophoresis (PFGE).

2. SCOPE: To provide the PulseNet participants with the same procedure for performing PFGE of
Yersinia pestis thus ensuring interlaboratory comparability of the generated results.

3. DEFINITIONS/TERMS:

3.1 PFGE: Pulsed-field Gel Electrophoresis

3.2 DNA: Deoxyribonucleic acid
3.3 CDC: Centers for Disease Control and Prevention
3.4 CLRW: Clinical Laboratory Reagent Water

4. RESPONSIBILITIES/PROCEDURE:

PREPARATION OF PFGE PLUGS FROM AGAR CULTURES

BIOSAFETY WARNING: Yersinia pestis is a human pathogen and can cause serious disease. Check with your
instituation’s select agent policy before handling these isolates. Biosafety Level 2 or 3 (depending on your instiution) are
required when handling this agent. Always use extreme caution when transferring and handling strains in general. Work
in a biological safety cabinet when handling large amounts of cells. Disinfect or dispose of all plasticware and glassware
that come in contact with the cultures in a safe manner.

Please read all instructions carefully before starting protocol. Treat all plasticware, glassware, pipets, spatulas, etc. that
come in contact with the cell suspensions or plugs as contaminated materials and dispose of, or disinfect according to the
guidelines of your institution. Disinfect reusable plug molds before they are washed; the disposable plug molds,
including the tape and the tab that is used to push the plugs out of the wells, are also contaminated and should be
disinfected with 10% bleach for at least 30 minutes if they will be washed and reused.

Day 0
Streak an isolated colony from test cultures to a Blood agar plate fortified with 6% sheep blood (or comparable media)
for confluent growth. Incubate cultures at 28°C (room temp) or 37°C for 48 h. If additional diagnostic tests for the
presence of F1 antigen are to be done on the isolate, cultures need to be incubated at 37°C for the F1 antigen to be
expressed.

Day 1
1. Turn on shaker water bath (54ºC), stationary water baths (55-60ºC) and spectrophotometer (or equivalent
instrument such as the Dade Microscan Turbidity meter or bioMérieux Vitek colorimeter).

2. Prepare TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) 1 as follows:

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure Clinical Laboratory Reagent Water (CLRW)

1
Additional information is found in the PulseNet PFGE Manual.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
5 25 2007

Note: The TE Buffer is used to make the plug agarose and also to wash lysed PFGE plugs.
3. Prepare 1% SeaKem Gold:1% SDS agarose in TE Buffer (10 mM Tris:1 mM EDTA,
pH 8.0) for PFGE plugs as follows:

a. Weigh 0.50 g (or 0.25 g) SeaKem Gold (SKG) into 250 ml screw-cap flask.
b. Add 47.0 ml (or 23.5 ml) TE Buffer; swirl gently to disperse agarose.
c. Remove cap, cover loosely with clear film, and microwave for 30-sec; mix gently and repeat for 10-sec
intervals until agarose is completely dissolved. Place flask in 55-60ºC water bath for 5 minutes before
adding SDS.
d. Add 2.5 ml (or 1.25 ml) of 20% SDS (pre-heated to 55ºC) and mix well.
e. Recap flask and return to 55- 60ºC water bath until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: SeaKem Gold agarose works well for making PFGE plugs because it provides added strength to the plugs that are
cast in reusable plug molds, minimizing breakage of plugs during the lysis and washing steps. The time and temperature
needed to completely dissolve the agarose is dependent on the specifications of the microwave used, and will have to be
determined empirically in each laboratory.

4. Label small tubes (12-mm x 75-mm Falcon tubes or equivalent) with culture numbers.

5. Prepare Cell Suspension Buffer (100 mM Tris:100 mM EDTA, pH 8.0) as follows:

10 ml of 1 M Tris, pH 8.0
20 ml of 0.5 M EDTA, pH 8.0
Dilute to 100 ml with sterile Ultrapure water (CLRW)

6. Transfer ≈2 ml of Cell Suspension Buffer (CSB) to small labeled tubes. Use a sterile polyester-fiber or cotton swab
that has been moistened with sterile CSB to remove some of the growth from agar plate; suspend cells in CSB by
spinning swab gently so cells will be evenly dispersed and formation of aerosols is minimized.

Note: The minimum volume of the cell suspension needed will depend on size of the cuvettes or tubes used to measure
the cell concentration and are dependent on the manufacturer’s specifications for the spectrophotometer, turbidity meter,
or colorimeter. Keep suspensions on ice if you have more than 6 cultures to process or refrigerate cell suspensions if you
cannot adjust their concentration immediately.

7. Adjust concentration of cell suspensions to one of values given below by diluting with sterile CSB or by adding
additional cells.

a. Spectrophotometer: 610 nm wavelength, absorbance (Optical Density) of 1.35

(range of 1.3-1.4)

b. Dade Microscan Turbidity Meter: 0.48 - 0.53 (measured in Falcon 2054 tubes)
0.68 - 0.72 (measured in Falcon 2057 tubes)

c. bioMérieux Vitek colorimeter: 20% transmittance (measured in Falcon 2054 tubes)

Note: Cell suspensions need to be at room temperature when concentration is checked. The values in Steps 7a. and 7b.
give satisfactory results at CDC; if different instruments or tubes are used, each laboratory may need to establish the
concentration needed for satisfactory results.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
5 25 2007

CASTING PLUGS

Label wells of PFGE plug molds with culture number. When reusable plug molds are used, put strip of tape on lower
part of reusable plug mold before labeling wells.

Note 1: Unused plug agarose can be kept at room temperature and reused 1-2 times. Microwave on low-medium power
for 10 -15 sec and mix; repeat for 5 -10 sec intervals until agarose is completely melted. This agarose melts rapidly!

Note 2: Proteinase K solutions (20 mg/ml) are available commercially, or a stock solution of Proteinase K can be
prepared from the powder in sterile Ultrapure water (CLRW), aliquoted in 300-500 μl amounts, and kept frozen. Just
before use, thaw appropriate number of vials needed for the samples; keep Proteinase K solutions on ice. Discard any
thawed Proteinase K stock solution that was prepared from powder by the user at end of work day. Store commercially
prepared Proteinase K solutions according to directions provided by the supplier.

1. Transfer 400 μl (0.4 ml) adjusted cell suspensions to labeled 1.5-ml microcentrifuge tubes. If cell suspensions
are at room temperature, agarose can be added directly without pre-warming cell suspensions. If cell
suspensions are cold, place tubes containing cell suspensions in plastic holders (floats); incubate in a 37ºC
water bath for a few minutes.

2. Add 20 μl of Proteinase K (20 mg/ml stock) to each tube and mix gently with pipet tip. (200 μl are needed for
10 cell suspensions.)

3. Add 400 μl (0.4 ml) melted 1% SeaKem Gold:1% SDS agarose to the 0.4-ml cell suspension; mix by gently
pipetting mixture up and down a few times. Maintain temperature of melted agarose by keeping flask in beaker
of warm water (55-60ºC).

4. Immediately, dispense part of mixture into appropriate well(s) of disposible plug mold.* Do not allow bubbles
to form. Two plugs of each sample can be made from these amounts of cell suspension and agarose. Allow
plugs to solidify at room temperature for 10-15 min. They can also be placed in the refrigerator (4ºC) for 5
minutes.

* Disposible plug molds are recommended for BSL-3 work as the plug molds can be discarded easily.
Note: If disposable plug molds are used for making plugs with 1% SeaKem Gold:1% SDS agarose, use 200 μl cell
suspension, 10 μl of Proteinase K (20 mg/ml stock) and 200 μl of agarose; up to 4 plugs can be made from these amounts
of cell suspension and agarose.

LYSIS OF CELLS IN AGAROSE PLUGS

Note: Two plugs (reusable plug molds) or 3 - 4 plugs (disposable plug molds) of the same strain can be lysed in the same
50-ml tube.

1. Label 50-ml polypropylene screw-cap or 50-ml Oak Ridge tubes with culture numbers.

2. Prepare Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) as follows:
25 ml of 1 M Tris, pH 8.0
50 ml of 0.5 M EDTA, pH 8.0

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

50 ml of 10 % Sarcosyl (N-Lauroylsarcosine, Sodium salt) 2

Dilute to 500 ml with sterile Ultrapure water (CLRW)

3. Calculate the total volume of Cell Lysis/Proteinase K Buffer needed as follows:

a. 5 ml Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) is needed per tube
(e. g., 5 ml x 10 tubes = 50 ml).

b. 25 μl Proteinase K stock solution (20 mg/ml) is needed per tube of the cell lysis buffer
(e. g., 25 μl x 10 tubes = 250 μl).

c. Measure correct volumes into appropriate size test tube or flask and mix well.

Note: The final concentration of Proteinase K in the lysis buffer is 0.1 mg/ml, and is different from the concentration
that was added to the cell suspension (0.5 mg/ml).

4. Add 5 ml of Proteinase K/Cell Lysis Buffer to each labeled 50 ml tube.

5. Trim excess agarose from top of plugs with scalpel or razor blade (optional). Open reusable plug mold and transfer
plugs from mold with a 6-mm wide spatula to appropriately labeled tube.
If disposable plug molds are used, remove white tape from bottom of mold and push out plug(s) into appropriately
labeled tube. Be sure plugs are under buffer and not on side of tube.

Note: The excess agarose, plug mold, spatula, etc. are contaminated. Discard or disinfect appropriately.

6. Remove tape from reusable mold. Place both sections of plug mold, spatulas, and scalpel in 70% isopropanol
(IPA) or other suitable disinfectant. Soak them for 15 minutes before washing them. Discard disposable plug
molds or disinfect them in 10% bleach for 30-60 minutes if they will be washed and reused.

7. Place tubes in rack and incubate in a 54ºC shaker water bath for 2 h with constant and vigorous agitation (175-200
rpm). Be sure water level in water bath is above level of lysis buffer in tubes.

8. Pre-heat enough sterile Ultrapure water (CLRW) to 50ºC so that plugs can be washed two times with 10-15 ml water
(200-250 ml for 10 tubes).

WASHING OF AGAROSE PLUGS AFTER CELL LYSIS

Lower the temperature of the shaker water bath to 50ºC.

1. Remove tubes from water bath, and carefully pour off lysis buffer into an appropriate discard container; plugs can be
held in tubes with a screened cap or spatula.

Note: It is important to remove all of the liquid during this and subsequent wash steps by touching edge of tube or
screened cap on an absorbent paper towel.

2
The N-Lauroylsarcosine, Sodium salt can be added directly to the other ingredients and allowed to dissolve.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

2. Add at 10-15 ml sterile Ultrapure water (CLRW) that has been pre-heated to 50ºC to each tube and shake the tubes
vigorously in a 50ºC water bath for 10-15 min.

3. Pour off water from the plugs and repeat wash step with pre-heated water (Step 2) one more time.
a. Pre-heat enough sterile TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) in a 50ºC water bath so that plugs can
be washed four times with 10-15 ml TE (300-350 ml for 10 tubes) after beginning last water wash.

4. Pour off water, add 10-15 ml pre-heated (50ºC) sterile TE Buffer, and shake the tubes vigorously in 50ºC water bath
for 10-15 min.

5. Pour off TE and repeat wash step with pre-heated TE three more times.

6. Decant last wash and add 5-10 ml sterile TE. Continue with step 1 in "Restriction Digestion" section or store plugs in
TE Buffer at 4ºC until needed. Plugs can be transferred to smaller tubes for storage.

Note: If restriction digestion is to be done the same day, complete Steps 1-3 of next section (RESTRICTION
DIGESTION OF DNA IN AGAROSE PLUGS WITH AscI & XbaI) during last TE wash step for optimal use of
time.

RESTRICTION DIGESTION OF DNA IN AGAROSE PLUGS WITH AscI & XbaI

Note: A small slice of the plug or the entire plug (made in disposable plug molds) can be digested with the restriction
enzyme. Restriction digestion of a small slice of the plug is recommended because less enzyme is required and other
slices of the plug can be subjected to restriction analysis with other enzymes, such as FseI, etc. This is important when
the PFGE patterns obtained with the primary enzyme from two or more isolates are indistinguishable, and confirmation
is needed to determine that the PFGE patterns of these isolates are also indistinguishable with additional enzymes.

1. Label 1.5-ml microcentrifuge tubes with Yersinia pestis culture numbers; label 3 (10-well gel) or 4 (15-well gel)
tubes for Salmonella ser. Braenderup H9812 standards.
a. Optional Pre-Restriction Incubation Step: Dilute 10X H buffer (Roche Molecular Biochemicals or
equivalent) and 10X Buffer 4 (New England Biolabs or equivalent) 1:10 with sterile Ultrapure water (CLRW)
according to the following table.

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical
Laboratory Reagent 180 μl 1800 μl 2700 μl
Water (CLRW)
H Buffer 20 μl 200 μl 300 μl

Total Volume 200 μl 2000 μl 3000 μl

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical
Laboratory Reagent 180 μl 1800 μl 2700 μl
Water (CLRW)
Buffer 4 20 μl 200 μl 300 μl

Total Volume 200 μl 2000 μl 3000 μl

b. Add 200 μl diluted H buffer (1X) to S. ser. Braenderup labeled 1.5-ml microcentrifuge tubes.

c. Add 200 ul diluted buffer 4 (1X) to Yersinia pestis labeled 1.5 ml microcentriufge tubes.

d. Carefully remove plug from TE with spatula and place in a sterile disposable Petri dish or on large glass slide.

e. Cut a 2.0- to 2.5-mm-wide slice from test samples with a single edge razor blade (or scalpel, cover slip, etc.)
and transfer to tube containing diluted buffer 4. Be sure plug slice is under buffer. Replace rest of plug in
original tube that contains 5 ml TE buffer. Store at 4ºC.

Note: The shape and size of the plug slice that is cut will depend on the size of the comb teeth that are used for casting
the gel. PulseNet recommends that the combs with larger teeth (10-mm-wide teeth) be used to cast the gels because
computer analysis of the gel lanes is more accurate and less tedious than analysis of gel lanes cast with combs with the
smaller teeth (5.5-mm). The number of slices that can be cut from the plugs will depend on the skill and experience of
the operator, integrity of the plug, and whether the slices are cut vertically or horizontally (plugs made in disposable
molds).

f. Cut three or four 2.0-mm-wide slices from plug of the S. ser. Braenderup H9812 standard and transfer to tubes
with diluted H buffer. Be sure plug slices are under buffer. Replace rest of plug in original tube that contains 5
ml TE buffer. Store at 4ºC.

g. Incubate sample and control plug slices in 37ºC water bath for 5-10 min or at room temperature for 10-15 min.

h. After incubation, remove buffer from plug slice using a pipet fitted with 200-250 μl tip all the way to bottom of
tube and aspirate buffer. Be careful not to cut plug slice with pipet tip and that plug slice is not discarded with
pipet tip.

2. Dilute 10X H buffer 1:10 with sterile Ultrapure water (CLRW) and add restriction enzyme 3 (50 U/sample for S. ser.
Braenderup and 40U/sample for Y. pestis) according to the following table. Mix in the same tube that was used for
the diluted H buffer.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

Reagent μl/Plug Slice Μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical
Laboratory Reagent 175 μl 1750 μl 2625 μl
Water (CLRW)
H Buffer 20 μl 200 μl 300 μl
Enzyme XbaI (10 U/μl) 5 μl 50 μl 75 μl

Total Volume 200 μl 2000 μl 3000 μl

Reagent μl/Plug Slice Μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical
Laboratory Reagent 174 μl 1740 μl 2610 μl
Water (CLRW)
Buffer 4 20 μl 200 μl 300 μl
Bovine Serum Albumin
2 ul 20 ul 30 ul
(BSA)
Enzyme AscI (10 U/μl) 4 μl 40 μl 60 μl

Total Volume 200 μl 2000 μl 3000 μl

Note: Keep vial of restriction enzyme on ice or in insulated storage box (-20˚C) at all times.

3. Add 200 μl restriction enzyme mixture to each tube. Close tube and mix by tapping gently;
be sure plug slices are under enzyme mixture.

4. Incubate sample and control plug slices in 37˚C water bath for 4 h.

5. If plug slices will be loaded into the wells, continue with Steps 1-4 of the next section (CASTING AGAROSE
GEL) approximately 1 h before restriction digest reaction is finished so the gel can solidify for at least 30 minutes
before loading the restricted PFGE plugs.

CASTING AGAROSE GEL

A. Loading Restricted Plug Slices on the Comb:

1. Confirm that water bath is equilibrated to 55- 60ºC.

2. Make volume of 0.5X Tris-Borate EDTA Buffer (TBE) that is needed for both the gel and electrophoresis running
buffer according to one of the following tables.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

5X TBE:
Reagent Volume in milliliters (ml)
5X TBE 200 210 220 230 240 250
Sterile Clinical Laboratory
1800 1890 1980 2070 2160 2250
Reagent Water (CLRW)
Total Volume of 0.5X TBE 2000 2100 2200 2300 2400 2500

10X TBE:
Reagent Volume in milliliters (ml)
10X TBE 100 105 110 115 120 125
Sterile Clinical Laboratory
1900 1995 2090 2185 2280 2375
Reagent Water (CLRW)
Total Volume of 0.5X TBE 2000 2100 2200 2300 2400 2500

3. Make 1% SeaKem Gold (SKG) Agarose in 0.5X TBE as follows:

a. Weigh appropriate amount of SKG into 500 ml screw-cap flask.

b. Add appropriate amount of 0.5X TBE; swirl gently to disperse agarose.
c. Remove cap, cover loosely with clear film, and microwave for 60-sec; mix
gently and repeat for 15-sec intervals until agarose is completely dissolved.
d. Recap flask and place in 55-60ºC water bath.

Mix 1.0 g agarose with 100 ml 0.5X TBE for 14-cm-wide gel form (10 wells)
Mix 1.5 g agarose with 150 ml 0.5X TBE for 21-cm-wide gel form (≥15 wells)

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

4. A small volume (2-5 ml) of melted and cooled (50-60ºC) 1% SKG 1% SKG agarose may be wanted to seal wells
after plugs are loaded. Prepare 50 ml by melting 0.5 g agarose with 50 ml 0.5X TBE in 250 ml screw-cap flask as
described above. Unused SKG agarose can be kept at room temperature, melted, and reused several times.
Microwave for 15-20 sec and mix; repeat for 10-sec intervals until agarose is completely melted. Place in 55-60ºC
water bath until ready to use. Alternatively, save approximately 5 ml of the melted agarose used to cast the gel in a
pre-heated (55-60ºC) 50 ml flask and place in 55-60ºC water bath until used.

Note: Confirm that gel form is level on leveling table, that front of comb holder and teeth face the bottom of gel, and
that the comb teeth touch the gel platform.

5. Remove restricted plug slices from 37ºC water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE.
Incubate at room temperature for 5 min.

6. Remove plug slices from tubes; put comb on bench top and load plug slices on the bottom of the comb teeth as
follows:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

a. Load S. ser. Braenderup H9812 standards on teeth (lanes) 1, 5, 10 (10-well gel) or on teeth 1, 6, 10, 15 (15-well
gel)
b. Load samples on remaining teeth

7. Remove excess buffer with tissue. Allow plug slices to air dry on the comb for ≈5 minutes or seal them to the
comb with 1% SKG agarose (55-60ºC).

8. Position comb in gel form and confirm that the plugs slices are correctly aligned on the bottom of the comb teeth,
that the lower edge of the plug slice is flush against the black platform, and there are no bubbles (if allowed to air
dry).

9. Carefully pour the agarose (cooled to 55-60ºC) into the gel form.

10. Put black gel frame in electrophoresis chamber. Add 2 -2.2 L freshly prepared 0.5X TBE. Close cover of unit.
(The amount of buffer needed depends on whether residual buffer was left in tubing or if unit was flushed with
water after the last gel was run.)

11. Turn on power supply to CHEF Mapper, pump (setting of ≈70 for a flow of
1 liter/minute) and cooling module (14°C).

12. Remove comb after gel solidifies for 20 minutes.

13. Fill in wells of gel with melted and cooled (55- 60ºC) 1% SKG Agarose (optional). Unscrew and remove end
gates from gel form; remove excess agarose from sides and bottom of casting platform with a tissue. Keep gel on
casting platform and carefully place gel inside black gel frame in electrophoresis chamber. Close cover of
chamber.

B. Loading Restricted Plug Slices into the Wells:

1. Follow steps 1-4 in Option A on pages 7 and 8 (Loading Restricted Plug Slices on the Comb).

Note: Confirm that gel form is level on gel-leveling table before pouring gel, that front of comb holder and teeth face
bottom of gel, and the bottom of the comb is 2 -mm above the surface of the gel platform.

2. Cool melted SKG agarose in 55-60ºC water bath for 15-20 min; carefully pour agarose into gel form (casting
stand) fitted with comb. Be sure there are no bubbles.

3. Put black gel frame in electrophoresis chamber. Add 2-2.2 L freshly prepared 0.5X TBE. Close cover of unit. (The
amount of buffer depends on whether residual buffer was left in tubing, or if unit was flushed with water after the
last gel was run.)

4. Turn on power supply to CHEF Mapper, pump (setting of ≈70 for a flow of
1 liter/minute) and cooling module (14°C).

5. Remove restricted plug slices from 37ºC water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE.
Incubate at room temperature for 5 minutes.

6. Remove comb after gel solidifies for at least 20 minutes.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

7. Remove restricted plug slices from tubes with tapered end of spatula and load into appropriate wells. Gently push
plugs to bottom and front of wells with wide end of spatula. Manipulate position with spatula and be sure that are
no bubbles.

a. Load S. ser. Braenderup H9812 standards in wells (lanes) 1, 5, 10 (10-well gel) or in wells 1, 6, 10, 15 (15-
well gel).

b. Load samples in remaining wells.

Note: Loading the plug slices can be tedious; each person has to develop his/her own technique for consistently placing
the plug slices in the wells so the lanes will be straight and the bands sharp.

8. Fill in wells of gel with melted 1% SKG Agarose (equilibrated to 55- 60ºC). Allow to harden for 3-5 min.
Unscrew and remove end gates from gel form; remove excess agarose from sides and bottom of casting platform
with a tissue. Keep gel on casting platform and carefully place gel inside black gel frame in electrophoresis
chamber. Close cover of chamber.

ELECTROPHORESIS CONDITIONS

1a. Select following conditions on Chef Mapper for Yersinia pestis restricted with AscI
Auto Algorithm
25 kb - low MW
215 kb - high MW
Select default values except where noted by pressing "enter".
Change run time to 17:30 h (See note below)
(Default values: Initial switch time = 1.79 s; Final switch time = 18.66 s)
linear ramping factor

1b. Select following conditions on CHEF DR II or III.

Initial A time: 1.79 s
Final A time: 18.66 s
Start ratio: 1.0
Voltage: 200 V
Run time: 20-22 h (DR II); 18-20 h (DR III)

Note: The electrophoresis running times recommended above are based on the equipment and reagents used at the CDC.
Run times may be different in your laboratory and will have to be optimized for your gels so that the lowest band in the
S. ser. Braenderup H9812 standard migrates 1.0 - 1.5 cm from the bottom of the gel.

Day 2

STAINING AND DOCUMENTATION OF PFGE AGAROSE GEL

1. When electrophoresis run is over, turn off equipment (cooling module FIRST then pump); remove and stain gel
with ethidium bromide. Dilute 40 μl of ethidium bromide stock solution (10 mg/ml) with 400 ml of Ultrapure
water (CLRW) (this volume is for a staining box that is approximately 14-cm x 24-cm; a larger container may
require a larger amount of staining solution). Stain gel for 20 - 30 min in covered container.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

Note: Ethidium bromide is toxic and a mutagen; the solution can be kept in dark bottle and reused 4 - 5 times before
discarding according to your institution's guidelines for hazardous waste or use the destaining bags recommended for
disposal of ethidium bromide (Section 10).

2. Destain gel in approximately 500 ml Ultrapure water (CLRW) for 60 - 90 min; change water every 20 minutes.
Capture image on Gel Doc 1000, Gel Doc 2000, or equivalent documentation system. If background interferes
with resolution, destain for an additional 30-60 min.

Note: If both a digital image and conventional photograph are wanted, photograph gel first before capturing digital
image.

3. Follow directions given with the imaging equipment to save gel image as an *.img or *.1sc file; convert this file to
*.tif file for analysis with the BioNumerics software program (Additional information is in Section 11 of the PFGE
Manual).

4. Drain buffer from electrophoresis chamber and discard. Rinse chamber with 2 L Ultrapure water (CLRW) or, if
unit is not going to be used for several days, flush lines with water by letting pump run for 5-10 min before
draining water from chamber.

Please note the following if PFGE results do not have to be available within 24-28 hours:

1. Plugs can be lysed for longer periods of time (3-16 hours).

2. The washing steps with TE to remove the lysis buffer from the PFGE plugs can be done for longer periods of time
(30-45 min) and at lower temperatures (37°C or room temperature). They can be started on Day 1 and finished on
Day 2 after overnight refrigeration of the plugs in TE.

3. The restriction digestion can be done for longer periods of time (3-16 hours).

4. If the lowest band in the H9812 standard does not migrate within 1 -1.5 cm of the bottom of the gel, the run time
will need to be determined empirically for the conditions in each laboratory.

Use of trade names and commercial sources is for identification purposes only and does not imply endorsement by CDC
or the U.S. Department of Health and Human Services.

NOTE: CLIA LABORATORY PROCEDURE MANUAL REQUIREMENTS

Efforts have been made to assure that the procedures described in this protocol have been written in accordance with the
1988 Clinical Laboratory Improvement Amendments (CLIA) requirements for a procedure manual (42 CFR 493.1211).
However, due to the format required for training, the procedures will require some modifications and additions to
customize them for your particular laboratory operation.

Any questions regarding the CLIA requirements for a procedure manual, quality control, quality assurance, etc., should
be directed to the agency or accreditation organization responsible for performing your laboratory's CLIA inspection. In
addition, some states and accreditation organizations may have more stringent requirements that will need to be
addressed.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

Formulas of Selected Reagents used in PulseNet Standardized Laboratory Protocol for PFGE

Tris:EDTA Buffer, pH 8.0 (TE, 10 mM Tris:1 mM EDTA, pH 8.0) 4

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure water (CLRW)

Cell Suspension Buffer (100 mM Tris:100 mM EDTA, pH 8.0)

10 ml of 1 M Tris, pH 8.0
20 ml of 0.5 M EDTA, pH 8.0
Dilute to 100 ml with sterile Ultrapure water (CLRW)

Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosine + 0.1 mg/ml Proteinase K)

25 ml (50 ml) of 1 M Tris, pH 8.0

50 ml (100 ml) of 0.5 M EDTA, pH 8.0
50 ml (100 ml) 10% N-Lauroylsarcosine, Sodium salt (Sarcosyl)
or
5 g (10 g) of N-Lauroylsarcosine, Sodium salt (Sarcosyl) 5
Dilute to 500 ml (1000 ml) with sterile Ultrapure water (CLRW)

Add 25 μl Proteinase K stock solution (20 mg/ml) per 5 ml of cell lysis buffer just before use
for a final concentration in the lysis buffer of 0.1 mg/ml Proteinase K.

Additional enzyme for Y pestis PFGE

Use the following calculations for FseI (40 Units/plug slice):

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory
163 μl 1630 μl 2445 μl
Reagent Water (CLRW)
Buffer 4 20 μl 200 μl 300 μl
BSA 2 µl 20 μl 30 μl
Enzyme FseI (10 U/μl) 15 μl 150 μl 225 μl

Total Volume 200 μl 2000 μl 3000 μl

4
This formula for TE is from Molecular Cloning - A Laboratory Manual by J. Sambrook and E. Russell, 3rd edition.
TE Buffer from Life Technologies (CP0558; 0126A) used at CDC is 0.01M (10 mM) for both ingredients.
To duplicate this commercial formula, increase the amount of 0.5 M EDTA to 20 ml per liter.
5
If Sarcosyl powder is added directly to the other components of this reagent, warm the solution to 50- 60ºC
for 30-60 minutes, or leave at room temperature for ≈2 hours to completely dissolve the Sarcosyl; adjust to the
final volume with sterile Ultrapure Water.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 13
 CODE: PNL18
STANDARD OPERATING PROCEDURE FOR PFGE OF YERSINIA PESTIS Effective Date:
4 03 2006

Note: Keep vial of restriction enzyme on ice or in insulated storage box (-70ºC) during storage. Keep FseI on ice or in
an insulated storage box at all times when in use.

ELECTROPHORESIS CONDITIONS

1a. Select following conditions on Chef Mapper for Yersinia pestis restricted with FseI
Auto Algorithm
30 kb - low MW
286 kb - high MW
Select default values except where noted by pressing "enter".
Change run time to 17:30 h (See note below)
(Default values: Initial switch time = 2.16 s; Final switch time = 25.0 s)
linear ramping factor

1b. Select following conditions on CHEF DR II or III.

Initial A time: 2.16 s
Final A time: 25.0 s
Start ratio: 1.0
Voltage: 200 V
Run time: 20-22 h (DR II); 18-20 h (DR III)

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:
8.1 The phrase “Type I Water” has been changed to “Ultrapure Clinical Laboratory Reagent Water
(CLRW).” The water composition is the same, but this reflects a change in the terminology used by
the Clinical Laboratory Standards Institute (CLSI).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 13
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

1. PURPOSE: to describe the standardized laboratory protocol for molecular subtyping of Shiga
toxin-producing Escherichia coli O157 (STEC O157) and Salmonella enterica serotypes
Typhimurium and Enteritidis.

2. SCOPE: to provide the PulseNet participants with a single protocol for performing MLVA of
STEC O157 and Salmonella serotypes Typhimurium and Enteritidis, thus ensuring inter-
laboratory comparability of the generated results using the Beckman Coulter CEQ
8000/8800/GeXP platform.

3. DEFINITIONS:
3.1. MLVA: Multiple-locus variable-number tandem repeat analysis
3.2. VNTR: Variable-number tandem repeat
3.3. DNA: Deoxyribonucleic acid
3.4. DNase: Deoxyribonuclenase
3.5. PCR: Polymerase chain reaction
3.6. HPLC: High purity liquid chromatography
3.7. dNTP: Deoxyribonucleotide triphosphate
3.8. CDC: Centers for Disease Control and Prevention

4. RESPONSIBILITIES/PROCEDURE
4.1. Biosafety warning: STEC O157 and Salmonella serotypes Typhimurium and Enteritidis
with an infectious dose as low as 100 cells are human pathogens capable of causing
serious disease. Always use a minimum of Biosafety level 2 practices and extreme
caution when transferring and handling strains of these serotypes. Work in a biological
safety cabinet when handling large amounts of cells. Disinfect or dispose of all plastic
ware and glassware that come in contact with the cultures in a safe manner.
4.2. Reagents, supplies and equipment needed for DNA template preparation
4.2.1. Trypticase soy agar with 5 % sheep blood (TSA-SB) or comparable media
4.2.2. 1 µl inoculation loops
4.2.3. 0.5 ml microcentrifuge tubes
4.2.4. DNase-free, molecular biology -grade water
4.2.5. Vortex
4.2.6. Boiling water bath or thermal block / thermocycler accommodating 0.5 ml tubes
4.2.7. Tabletop centrifuge for high rpm (up to 13,000-14,000 rpm) spinning
4.3. Reagents, supplies and equipment needed for PCR
4.3.1. DNA templates from isolates (store at -20oC or -80ºC freezer for long term)
4.3.2. PCR primers (see appendix PNL19-1)
4.3.2.1. Fluorescent-labeled forward primers
4.3.2.1.1. HPLC-purified
4.3.2.2. Unlabeled reverse primers
4.3.2.2.1. Regular gel filtration purification
4.3.2.3. Integrated Technologies (Skokie, IL; www.idtdna.com, 1-800-
328-2661) and Proligo (Boulder, CO; www.proligo.com, 1-800-234-
5362) are currently the suppliers for the Wellred dye-labeled (D2)
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

primers recommended by Beckman Coulter. Biosearch Technologies

(Novato, CA; www.biosearchtech.com; 1-800-436-6631) is the
recommended supplier for the Quasar dye –labeled (Quas670 and
705) primers.
4.3.2.4. Divide the concentrated stocks (100 µM) in portions and store at
-80ºC freezer
4.3.2.4.1. One vial should contain enough to prepare 25-50 µl of
working solution. Avoid repetitive freeze-thaw cycles of
concentrated primer stocks.
4.3.2.5. The 1, 2.5, 5, 12.5 and 25 µM working solutions can be stored at
either -20oC or -80ºC freezer
4.3.2.6. Prepare new working solutions every month or if a significant
drop in the fluorescence level is observed (for instructions refer to
PNQ05_MLVA CEQ certification, appendix PNQ05-5)
4.3.3. 96-well polypropylene PCR plates (Fisher, Cat. No. 07-200-613) or Microamp
PCR tubes without caps (Life Technologies, Cat. No. N8010533)
4.3.4. 8-well strip caps for polypropylene plate (Fisher, Cat. No. 07-200-639) or
MicroAmp strip caps for individual tubes (Life Technologies, Cat. No.
N8010535)
4.3.5. DNase-free, molecular biology -grade water
4.3.6. 1.5 ml Eppendorf microcentrifuge tubes
4.3.7. PCR Nucleotide mix (ready-to-use dNTP mix containing all four nucleotides;
Roche, Cat. No. 11 814 362 001)
4.3.8. Platinum Taq Polymerase with 50 mM MgCl2 and 10X buffer (Life
Technologies, Cat. No. 10966-034)
4.3.9. PCR cooling block (VWR International, Cat. No. 62111-762)
4.3.10. DNA Engine (Biorad), GeneAmp (Life Techologies) or similar thermocycler
with a heated lid option and 96-well block format
4.3.11. Parafilm M, 4” width (VWR, Cat. No. 52858-032)
4.3.12. Complete set (1000 µl, 200 µl, 100 µl, 20 µ, 10 µl, and 2 µl) of single channel
pipettors for mastermix set-up (“clean set”)
4.3.13. A 1-10 µl single channel pipettor for adding DNA templates
4.3.14. Filtered tips for pipettors
4.3.15. Microfuge for low (up to 6,000 rpm) rpm spinning
4.4. Reagents, supplies and equipment needed for Beckman CEQ 8000/ 8800/GeXP)
4.4.1. DNase-free, molecular biology -grade water
4.4.2. PCR cooling block (VWR International, Cat. No. 62111-762)
4.4.3. 10 l, 100 l, and 1000 l single channel pipettors
4.4.4. 1-10 l and 20-200 µl multichannel pipettors
4.4.5. Filtered pipette tips
4.4.6. Sterile solution basins
4.4.7. 1.5 ml Eppendorf microcentrifuge tubes
4.4.8. 5 ml polystyrene round bottom tubes (for example Falcon tube 352058)

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

4.4.9. 96-well polypropylene (non-PCR) V-bottom plate (for dilutions; Fisher

Scientific, Cat. No. 07-200-698)
4.4.10. 96-well Beckman sample plates (Beckman Coulter, Cat. No. 609801;
alternative: Fisher Scientific, Cat. No. 07-200-613)
4.4.11. 96-well Beckman CEQ buffer plate (Beckman Coulter, Cat. No. 609844;
alternative: Fisher Scientific, Cat. No. 07-200-98)
4.4.12. Mineral oil (molecular grade) (Sigma, Cat. No. M5904)
4.4.13. GenomeLab Sample Loading Solution (Beckman Coulter, Cat. No. 608082)
4.4.13.1. After first thawing, divide in portions of 1.0 ml / vial in order to
avoid repeated freeze-thaw cycles
4.4.14. GenomeLab DNA size standard kit 600 bp (Beckman Coulter, Cat. No.
608095)
4.4.14.1. After first thawing, divide in portions of 8 µl / vial in order to
avoid repeated freeze-thaw cycles
4.4.15. GenomeLab Fragment Analysis Test Sample (Beckman Coulter, Cat. No.
608105)
4.4.15.1. Needed only once to establish the system dye color spectra for
the instrument
4.4.15.2. Useful also for troubleshooting
4.4.16. GenomeLab Separation Capillary Array (Beckman Coulter, Cat. No. 608087)
4.4.17. GenomeLab Separation Buffer, 4x30 ml (Beckman Coulter, Cat. No. 608012)
4.4.18. GenomeLab Separation Gel LPAI (Beckman Coulter, Cat. No. 608010 for
CEQ 8000/single rail GeXP; Cat. No. 391438 for CEQ 8800/double rail GeXP)
4.4.19. Parafilm M, 4” width (VWR, Cat. No. 52858-032)
4.4.20. A rinse bottle containing distilled water
4.4.21. Centrifuge with a microtiter plate rotor
4.4.22. Microfuge for low (up to 6,000 rpm) rpm spinning
4.5. Isolate preparation
4.5.1. Day 0
4.5.1.1. Streak an isolated colony from pure test cultures to TSA-SB
plate (or comparable media). Incubate cultures at 37ºC for 14-18 hrs.
4.5.2. Day 1
4.5.2.1. For each isolate to be typed, aliquot 100 µl of sterile, molecular
biology-grade water into 0.5 ml microfuge tubes. Use a sterile,
disposable 1 µl loop to pick 2-3 colonies (about half of a loopfull);
rotate the loop in the microfuge tube to release the bacteria into the
water. Cap and vortex for 10-15 seconds to disperse any clumps.
4.5.2.2. Place the tubes in a 99-100ºC water bath or heat block for 10-15
minutes. Cool briefly on ice or in fridge and centrifuge for 10
minutes at 10,000 rpm. Place on ice or in fridge while preparing PCR
reactions. These DNA templates can be stored at -20oC or -80ºC for
several years.
4.6. PCR procedure

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

4.6.1. Day 1
4.6.1.1. Fill out, save with the run name, and print a MLVA Fragment
Analysis CEQ Worksheet (see appendix PNL19-2) with
appropriately labeled samples (a maximum of 43 isolates/plate; 40
unknowns + two positive controls and a negative control; two wells
are reserved for the internal ladder; the first column of the plate is
reserved for conditioning).
4.6.1.1.1. For each isolate, two wells must be labeled as follows:
BNkeyR1 where “BNkey” represents the isolate-specific state
laboratory identification number (be sure to use the exact same
isolate ID that is used in the PFGE gels uploaded to the national
database) and “R1” represents one of the two specific multiplex
PCR reactions (R1, R2).
4.6.1.1.2. The CEQ8000/ 8800/GeXP machine has an 8-capillary
array and must always be run with a complete column (n=8
wells). For example, 54 wells (6.75 8-well columns) are required
to test 26 samples (including controls). The remaining two wells
for the seventh column must be filled and run with 20 µl of water
or left-over sample loading solution (blanks).
4.6.1.1.3. Also save the MLVA Fragment Analysis CEQ Worksheet for
the run on a flash drive (memory stick). That way you can easily
import the sample plate set-up in the CEQ without having to re-
type the sample keys.
4.6.1.2. Fill out, and print a PCR Mastermix Calculation Worksheet (see
appendices PNL19-3a, PNL19-3b, PNL19-3c) by typing the number
of isolates to be tested (plus 2-3 extra) in the PCR mastermix
calculators labeled R1 and R2. This number is highlighted in red and
is next to “number of samples to be analyzed”. The mastermixes for
reactions 1 and 2 (R1, R2) for one sample are as follows:

STEC O157:H7 (Appendix PNL19-3a)

R1 Volume ( µl) Final conc.

PCR water 5.16
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
VNTR-3F (25 µM) 0.18 0.45 µM
VNTR-3R (25 µM) 0.18 0.45 µM
VNTR-34F (5 µM) 0.28 0.14 µM
VNTR-34R (5 µM) 0.28 0.14 µM
VNTR-9F (5 µM) 0.28 0.14 µM
VNTR-9R (5 µM) 0.28 0.14 µM
VNTR-25F (2.5 µM) 0.28 0.07µM
VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

VNTR-25R (2.5 µM) 0.28 0.07 µM

Taq (5 U/µl) 0.20 1U
= 9.00

R2 Volume (µl) Final conc.

PCR water 5.96
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
VNTR-17F (5 µM) 0.18 0.09 µM
VNTR-17R (5 µM) 0.18 0.09 µM
VNTR-19F (1 µM) 0.20 0.02µM
VNTR-19R (1 µM) 0.20 0.02 µM
VNTR-36F (1 µM) 0.12 0.012 µM
VNTR-36R (1 µM) 0.12 0.012 µM
VNTR-37F (2.5 µM) 0.12 0.03µM
VNTR-37R (2.5 µM) 0.12 0.03µM
Taq (5 U/µl) 0.20 1U
= 9.00

Salmonella serotype Typhimurium (Appendix PNL19-3b)

R1 Volume (µl) Final conc.

PCR water 2.45
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.45 2.25 mM
dNTPs (10 mM) 0.20 0.20 mM
ST3-F (5 µM) 0.26 0.13 µM
ST3-R (5 µM) 0.26 0.13 µM
ST5-F (5 µM) 1.30 0.65 µM
ST5-R (5 µM) 1.30 0.65 µM
ST7-F (5 µM) 0.46 0.23 µM
ST7-R (5 µM) 0.46 0.23 µM
ST10-F (2.5 µM) 0.28 0.07 µM
ST10-R (2.5 µM) 0.28 0.07 µM
Taq (5 U/µl) 0.30 1.50 U
= 9.00

R2 Volume (µl) Final conc.

PCR water 0.06
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.32 1.60 mM
dNTPs (10 mM) 0.20 0.20 mM
ST2-F (5 µM) 1.60 0.80 µM
VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

ST2-R (5 µM) 1.60 0.80 µM

ST6-F4 (5 µM) 0.22 0.11 µM
ST6-R2 (5 µM) 0.22 0.11 µM
ST8-F3 (5 µM) 1.74 0.87 µM
ST8-R2 (5 µM) 1.74 0.87 µM
Taq (5 U/µl) 0.30 1.50 U
= 9.00

Salmonella serotype Enteritidis (Appendix PNL19-3c)

R1 Volume (µl) Final conc.

PCR water 4.41
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.0 mM
dNTPs (10 mM) 0.20 0.20 mM
SE1-F (1.0 µM) 0.30 0.03 µM
SE1-R (1.0 µM) 0.30 0.03 µM
SE2-F (12.5 µM) 0.14 0.17 µM
SE2-R (12.5 µM) 0.14 0.17 µM
SE8-F (2.5 µM) 0.28 0.07 µM
SE8-R (2.5 µM) 0.28 0.07 µM
SE6-F (2.5 µM) 0.68 0.17 µM
SE6-R (2.5 µM) 0.68 0.17 µM
Taq (5 U/µl) 0.20 1.00 U
= 9.00

R2 Volume (µl) Final conc.

PCR water 6.03
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
SE5-F (2.5 µM) 0.32 0.08 µM
SE5-R (2.5 µM) 0.32 0.08 µM
SE3-F (12.5 µM) 0.14 0.18 µM
SE3-R (12.5 µM) 0.14 0.18 µM
SE9-F (2.5 µM) 0.12 0.03 µM
SE9-R (2.5 µM) 0.12 0.03 µM
Taq (5 U/µl) 0.20 1.00 U
= 9.00
4.6.1.2.1. NOTE: NOTE: these primer concentrations serve as a starting
point. Since laboratory-specific factors, such as the age of the
primer stocks, calibration status of the thermocyclers and
pipettes, etc. affect amplification efficiency, each laboratory will
have to re-optimize the primer concentrations for optimal
VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

detection of all targets. However, any other parameters stated in

the SOP should not be changed.
4.6.1.3. Thaw all reagents and supplies needed for PCR reactions and
place on ice; keep primers light protected as much as possible.
4.6.1.3.1. PCR mastermixes should be set up in a clean hood that is
dedicated just for this purpose and where no cultures or DNA are
handled.
4.6.1.4. Prepare the two separate PCR mastermixes in 1.5 ml Eppendorf
tubes following the instructions in the PCR Mastermix Calculation
Worksheet (see appendix PNL19-3a-c) and place on ice. Add the
mastermix components in the following order: water, 10x PCR
buffer, Mg2Cl, dNTPs, primers, and then finally Taq polymerase.
Mix the reaction mixture by vortexing briefly.
4.6.1.4.1. Vortex all reagents except Taq polymerase before adding to
the mastermix. Taq may be briefly centrifuged with low rpm, if
necessary, to pull the enzyme down to the bottom of the tube.
4.6.1.5. Place a 96-well PCR plate or required number of PCR tubes in a
PCR cooling block.
4.6.1.6. Dispense 9.0 µl of each mastermix into the appropriate columns
of the 96-well polypropylene plate / PCR tubes as noted in the
MLVA Fragment Analysis CEQ Worksheet (see appendix PNL19-2)
4.6.1.7. Add 1 l of PCR water to each of the two different wells
representing the negative controls of the two reactions.
4.6.1.8. Add 1.0 l of DNA template to each of the two different wells
representing the two PCR reactions for each isolate to be tested.
4.6.1.9. Add the positive controls (it is recommended to run the positive
control in duplicate).
4.6.1.9.1. Use STEC O157 strain EDL933 (ATCC 43895) as a positive
control. The internal ladder to be used will be comprised of
pooled PCR products of the isolates EC04PN0139 and
EC04PN0570 (see appendix PNL19-4 for instructions for ladder
preparation).
4.6.1.9.2. Use S. enterica serotype Typhimurium strain LT2 (ATCC
29946) as a positive control. The internal ladder to be used will
be comprised of pooled PCR products of the isolates
CDC_2009K0825 and CDC_2009K0826 (see appendix PNL19-4
for instructions for ladder preparation).
4.6.1.9.3. Use S. enterica serotype Enteritidis strain K1891 (ATCC
25928) as a positive control. The internal ladder to be used will
be comprised of pooled PCR products of the isolates H9560 and
2010K0017 (see appendix PNL19-4 for instructions for ladder
preparation).
4.6.1.10. Cover all wells / tubes with 8-well strip caps and firmly clamp
down to avoid any evaporation during PCR amplification.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

4.6.1.10.1. Recommendation: briefly spin down the plate / tubes to

remove any air bubbles.
4.6.1.11. Program and save the following two PCR cycling conditions:

STEC O157:H7 and Salmonella serotype Enteritidis

“O157-SEMLVA”
* 95ºC for 5 min Step 1
* 94ºC for 20 sec Step 2
* 65ºC for 20 sec Step 3
* 72ºC for 20 sec Step 4
* Go to step 2, 34x Step 5
* 72ºC for 5 min Step 6
* Indefinite hold at 4ºC Step 7

Salmonella serotype Typhimurium

“STMLVA”
* 95ºC for 5 min Step 1
* 94ºC for 20 sec Step 2
* 63ºC for 20 sec Step 3
* 72ºC for 20 sec Step 4
* Go to step 2, 34x Step 5
* 72ºC for 5 min Step 6
* Indefinite hold at 4ºC Step 7

4.6.1.11.1. Make sure to use the heated lid option on the PCR block
and “calculated” or tube control (instead of block or probe control)
as a temperature control method.
4.6.1.12. When the PCR is complete, store the amplification products
light-protected at 4ºC until ready to run on CEQ/GeXP. If the
fragment analysis is not performed the same day, the plate should be
stored at -20oC or -80ºC. The PCR products are stable for
approximately one month, when stored frozen.
4.7. Initial setup of CEQ8000/ 8800/GeXP instrument
4.7.1. NOTE: Steps 4.7.2.-4.7.8. only need to be performed before the very first run
and every time a new working database is created.
4.7.2. In the main page of the CEQ software version 8.0 or 9.0 or the GeXP software
version 10.2, click on the “Fragments” icon. Select “Analysis” in the bar at the
top of the window to open a window called “Analysis Parameters”.
4.7.3. Select “DefaultFragmentAnalysisParameters” in the top drop-down menu and
click “Edit”. In the appearing “Edit Fragment Analysis Parameters” window,
select the tab “Analysis Method” and select “Size Standard 600” for size standard
and “Quartic” for model. Do not make any other changes.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

4.7.4. Click on “Save As” and name the analysis parameter as “MLVA600”.
4.7.5. Go back to the main page of the CEQ program and click on the “Set Up” icon.
Select “Create a New Sample Plate” and click “OK”.
4.7.6. In the “Sample Setup” window, check to be sure that the methods “Condition”
and “Frag-test” are present in the drop-down menu below each column. These
should be the instrument default methods.
4.7.7. If they are not present or to create the frag-test_pause method, highlight any of
the default methods and select “Method” tab from below. This will open a
“Method” window. Click on the “Edit” button in this open window and put in the
following parameters in the “CEQ Methods” table below.

CEQ Methods:

Condition Frag-test_pause
Capillary Temperature 35 35
Wait 0 Yes
Denature Temperature 90 90
Time 0 120 seconds
Pause Time NA 10 min
Inject Voltage 2.0 kV 2.0 kV
Time 30 seconds 15 seconds
Separation Voltage 7.5 kV 6.0 kV
Time 35 minutes 60 minutes
Pause 0 minutes 0 minutes

4.7.8. Be sure to save the new method with new file names. Each time a new CEQ
database is generated, these methods are not copied. However, if these methods
were in the machine as a default (Frag-test_pause is not), they are copied when a
new database is created.
4.8. CEQ8000/ 8800/GeXP instrument preparation before each run
4.8.1. Day 1
4.8.1.1. Make sure a capillary array is installed in the instrument. For
installation, follow the instructions in the appendix PNL19-5.
4.8.1.1.1. If the instrument is used frequently, there is no need to remove
the array after every run. Beckman Coulter guarantees the shelf
life of the array for up to 30 days (or up to 100 runs) at room
temperature. However, as long as the positive control and the
internal ladder fall within the sizing range indicated in the
appendices PNL19-2 and PNL19-4 and the fragment analysis
failure rate is less than 5 %, the same capillary array can be used
up to 45 to 50 days.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

4.8.1.2. To setup the plate run on the CEQ\GeXP, click on the “Set Up”
icon on the CEQ\GeXP main page and select “Create a New Sample
Plate” and click “OK”.
4.8.1.3. Using a USB flash drive, open the MLVA Fragment Analysis
CEQ Worksheet (PNL19-2) saved for this plate run and copy only
the 96-well format and paste it directly into the blank sample setup
window.
4.8.1.4. Under the first column of the plate format, select the method
“Condition” from the drop-down menu. Select the method “Frag-
test_pause” for the columns containing the samples.
4.8.1.5. Highlight all columns containing samples. At the bottom of the
screen, select the “Analysis” tab, check the box for “Perform
analysis”, and select the “MLVA600” parameter set from the drop-
down menu.
4.8.1.5.1. NOTE: The export tab does not contain the correct format
(CSV) and should be left unchecked.
4.8.1.6. Save the plate with a run name following the standardized
PulseNet naming system: Use the unique identifier code that was
assigned to your laboratory by PulseNet for the first two to four
letters of the file name. The next two spaces indicate the year and the
next four spaces indicate the month and the date the run was
performed. For example GA070426 is a run made at the GA Public
Health Laboratory on April 26th 2007. If several runs are performed
the same day, separate the file names by using sequential numbers,
for example GA070426-1, GA070426-2.
4.8.1.7. Close “Set Up” module.
4.8.1.8. Click on the “Run” icon in the main CEQ\GeXP window. The
“Run Control – CEQ System” window will appear.
4.8.1.9. If the instrument does not have a gel cartridge installed, the
“Instrument Warning Message” window will appear stating
“Instrument Status: Loading Gel Cartridge”. Click “OK”.
4.8.1.10. If the instrument already has a gel cartridge installed, the above
message does not appear. In this case, you can check the amount of
gel available by clicking on the “Life” tab on the left side of the “Run
Control – CEQ System” window. If a new gel cartridge needs to be
installed, select “Release Gel Cartridge” from the “Replenish” drop-
down menu or from the toolbar and wait for the CEQ\GeXP to
display a green “Go” signal box.
4.8.1.10.1. NOTE: A full plate uses approximately 9.0 ml of gel.
4.8.1.11. Open the gel access door, pull out the yellow plug or the old gel
cartridge and install the new gel cartridge. Select “Install Gel
Cartridge” from the “Replenish” drop-down menu or from the tool
bar. The “Install Gel Cartridge” dialog box will open. Type in the lot
number for the gel, click on “Set To New” to zero out the time on the
VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

instrument (if the cartridge is new) or type in the number of hours the
gel cartridge (previously used) has spent in the instrument. For the
GeXP, designate whether the gel cartridge is ‘New’ or ‘Used’ by
selecting the corresponding radial button. If installing a previously
used cartridge, enter the number of hours on instrument. When
finished click on “Done”.
4.8.1.12. Perform a manual purge by selecting “Manifold Purge” from the
“Direct Control” drop-down menu or by clicking on the funnel on the
direct control screen. A “Manifold Purge” dialog box will open. Set
0.1 ml as the purge volume and 2 as the number of purge cycles and
click on “Purge”.
4.8.1.12.1. NOTE: a manifold purge is only necessary when a new/used
gel cartridge is inserted. If using a cartridge already installed in
the instrument, a manifold purge is not necessary.
4.9. Fragment analysis sample preparation
4.9.1. Day 1
4.9.1.1. NOTE: The fragment analysis method is not organism specific
therefore; STEC O157 and Salmonella serotypes Typhiumurium and
Entertidis may be run on a single fragment analysis plate.
4.9.1.2. Thaw the CEQ sample loading solution (SLS), 600 bp DNA size
standard and internal ladder (see appendix PNL19-4 for preparation
instructions) and place on ice.
4.9.1.3. While the reagents are thawing, prepare a Beckman buffer plate
by filling a required amount of wells about ¾ full (~250 l) with
CEQ Separation Buffer.
4.9.1.4. Prepare a 96-well V-bottom plate for diluting the PCR reactions.
Using a 200 l multichannel pipettor and a solution basin, dispense
59 µl of molecular-grade water in the required number of wells.
4.9.1.5. Remove the plate / tubes with the PCR reactions from the
thermocycler. Briefly spin down the plate / tubes, if necessary. Use a
10 l multichannel pipettor to transfer 1 l of each PCR reaction
directly across to the corresponding set of wells in the dilution plate.
In order to avoid cross-contamination, remove the strip cap from just
one column at a time and recap the column before opening the next
one.
4.9.1.6. For the internal ladder, combine R1 and R2 PCR products from
the four PCR reactions of both internal ladder isolates into one tube
to end with a total of 40 µl. Mix well by pipetting up and down a few
times and add 3 l of internal ladder in two wells.
4.9.1.7. Using a 200 µl multichannel pipettor, mix the dilutions by
pipetting up and down a few times. Cover the plate with Parafilm and
put in the fridge or on ice.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

4.9.1.8. Prepare a fragment analysis master mix containing DNA size

standard and SLS buffer for the samples following the calculations
indicated in the table below. The fragment analysis mastermix
calculations can also be performed using the autocalculate box at the
bottom of the MLVA Fragment Analysis CEQ Worksheet (see
appendix PNL19-2). Vortex briefly and place on ice.

Reagents Frag. anal. mastermix

CEQ SLS buffer 20 µl x (# samples +3)=
CEQ 600 bp size standard 0.08 µl x (# samples +3)=

4.9.1.9. Place a Beckman 96-well sample plate in a cold block. Add two
drops of separation buffer to each well of the column 1 (conditioning
lane). Next, aliquot 20 l of the prepared fragment analysis
mastermix to the required number of wells. Cover the plate loosely
with Parafilm.
4.9.1.10. Using the 10 l multichannel pipettor, add 1 l of 1:60 diluted
PCR reactions to the appropriate columns in the Beckman sample
plate. Keep sliding the Parafilm sheet from column to column to keep
track of the sample order.
4.9.1.11. Briefly (15 s) spin down the CEQ Beckman sample plate to
remove any air bubbles.
4.9.1.12. Overlay the wells containing samples with 1 drop of mineral oil.
4.9.1.13. Select “Start Sample Plate” from the “Run” pull-down menu or
click on the “Run” button in the toolbar.
4.9.1.13.1. For the CEQ 8000, a “Select the Sample Plate to Run”
window will open. Select the plate layout that was saved above.
The “Confirm Configuration-(Sample Plate Name)” window will
pop up. Confirm that the plate layout matches with the lanes
highlighted in the plate configuration window and click on “Load
Plates”. When the “Access Plates” dialog box appears, click on
“Start”. Wait for the CEQ to display the green “Go” signal box.
4.9.1.13.2. For the GeXP, a “Sample Plate Run Confirmation” window
will open. This offers the option to designate operator’s name.
At the bottom of the window, select “Sample Plate Name” and
select the plate layout that was previously saved above. Confirm
that the plate layout matches with the lanes highlighted in the
plate configuration window and that the plate location is correct.
If running two plates, designate which plate to run first by
selecting the corresponding radial button. (This window also
offers the option to replenish the gel cartridge at this time by
selecting the “Install Gel Cartridge” button at the bottom of the
window and follow instructions as previously described in

VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

4.8.1.11). Click on “Load Plates”. When the “Access Plates”

dialog box appears, click on “Start”. Wait for the GeXP to
display the green “Go” signal box.
4.9.1.14. Lift the sample access cover. Rinse and refill wetting tray(s) with
dH2O. (If setting up on the GeXP the corresponding wetting tray for
each rail must be filled if running two plates.) Dry off outer tray,
clean off any dried deposits of gel from the lid and place the wetting
tray(s) back on machine.
4.9.1.15. Load the buffer plate(s) on the front tray and the sample plate(s)
on the back tray with notched corners top right. Close the sample
access cover. Click on “Load”. This will take you back to the
“Sample Plate Run Confirmation” window.
4.9.1.16. Once the instrument has loaded the plates, the “Start” button will
be highlighted and can be clicked to start the run.
4.9.1.17. Running one column (8 wells) takes about 105 min. A full plate
run takes 19½ h. At end of the run, the data will be automatically
analyzed and saved.
4.10. Data export from the Beckman Coulter CEQ 8000/ 8800/GeXP
4.10.1. Day 2
4.10.1.1. To view the analyzed data, click on the “Fragments” icon in the
main CEQ window and a window labeled “Study” will open up.
4.10.1.1.1. If the “Study” window does not pop up, select “New Study”
from the “File” drop-down menu.
4.10.1.2. Select the “Analyzed Results” button and then select the desired
plate run listed in the window and click “OK”. The “Select Result
Data” window will open.
4.10.1.3. In the “Select Result Data” window, select the “Plate View” tab
and the plate format will pop up. Select the desired wells for viewing
from the plate by either clicking on them individually or select a
whole column by clicking on the column number. Selected wells will
be highlighted. Complete the sample selection by clicking on the
“Finish” button. “Fragment Analysis – New Study” window will
open with all the selected wells listed under the “Results” section.
4.10.1.4. Check the fragment result data (the fluorescent peaks) for each
well by clicking on the well IDs individually.
4.10.1.4.1. Make sure that all VNTRs amplified in the positive control
and that the fragment sizes are within the range specified in the
appendix PNL19-2 and record the fragment sizes on the MLVA
Fragment Analysis CEQ Worksheet.
4.10.1.4.2. The size calling for the internal ladder should also be within
the range specified in the appendix PNL19-2 (or PNL19-4).
4.10.1.4.3. Write down any failed reactions on the MLVA Fragment
Analysis CEQ Worksheet. Make a note of non-specific peaks,

VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

primer-dimers, and atypical appearance of the DNA size standard

peaks.
4.10.1.4.4. If the molecular size standard does not run all the way till the
end (640 bp) or if it skips fragments, a reaction is considered “a
fragment analysis failure” and should be re-run.
4.10.1.4.5. Low fluorescence level background peaks can sometimes
interfere with data analysis in BioNumerics. They can be filtered
out by using the following procedure:
4.10.1.4.5.1 From the “Study explorer” field on the top left corner,
click on “Fragment list”. “Exclusion filter set” is located
in the right top corner.
4.10.1.4.5.2From the “Name” drop-down menu, select the “pk height
(rfu)” option.
4.10.1.4.5.3From the “Operators” drop-down menu select the “<”
option. Type in “1000” (or the fluorescence unit value you
have determined is adequate to filter out the background
without eliminating the true positive peaks) in the
“Value(s)” field.
4.10.1.4.5.4Click on “Apply”.
4.10.1.5. Save the study: from “File” drop-down menu, select the option
“Save Study As”. “Save Study As” dialog box will appear. Name the
study with the standardized run name (for example, “GA070426”)
and click “OK”. This ensures that you don’t have to re-analyze the
samples if you want to go to check the raw data again later.
4.10.1.6. From the “File” drop-down menu, select the option “Export
Fragments/Genotypes”. “Export Fragments/Genotypes” dialog box
will appear. Choose the location you would like the file to be saved to
(examples: E:/Compact Disk or F:/Removable Disk). Name the peak
file containing the fragment list with the run name (see section
4.8.1.6. for instructions; for example “GA070426”), make sure that
the file type is “CSV (comma limited)” and save it by clicking on
“Save” button. “Export Status” dialog box will appear. Once the
export is completed, “OK” button will be highlighted and can be
clicked.
4.10.1.7. The remaining gel can stay in the instrument if it is going to be
used within 48 hours. The shelf-life of the gel at the room
temperature is 72 hours. Using an expired gel may cause inaccurate
sizing of fragments in some loci.

5. FLOW CHART:

6. BIBLIOGRAPHY:
6.1. Hyytiä-Trees, E., Smole, S. C., Fields, P. I.., Swaminathan, B., and Ribot, E. M. (2006)
Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-
VERSION: REPLACED BY: AUTHORIZED BY:

Page 14 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

number tandem repeat analysis (MLVA) of Shiga toxin-producing Escherichia coli O157
(STEC O157). Foodborne Pathog. Dis. 3, 118-131.
6.2. Hyytia-Trees, E., Lafon, P., Vauterin, P., and Ribot, E. (2010) Multi-laboratory
validation study of standardized multiple-locus VNTR analysis (MLVA) protocol for
Shiga toxin-producing Escherichia coli O157 (STEC O157): a novel approach to
normalize fragment size data between capillary electrophoresis platforms. Foodborne
Path. Dis. 7, 129-136.

7. CONTACTS:
7.1 Eija Trees, D.V.M., Ph.D.
PulseNet Next Generation Subtyping Methods Unit, EDLB, DFWED, CDC
(404) 639-3672
[email protected]
7.2 Patti Lafon
PulseNet Next Generation Subtyping Methods Unit, EDLB, DFWED, CDC
(404) 639-2828
[email protected]
7.3 Ashley Sabol
PulseNet Next Generation Subtyping Methods Unit, EDLB, DFWED, CDC
(404) 639-2947
[email protected]

8. AMENDMENTS:

1/27/2009: Separation buffer is to be used for conditioning instead of distilled water (step 4.9.1.8)
10/6/2009: statement was added to the step 4.10.1.7. that using expired gel may cause inaccurate
fragment sizing.
6/8/2011: Instructions for operating the new GeXP version of the Beckman Coulter
Genetic Analysis system were added.
4/10/2012: “Frag-test” CEQ running conditions were replaced with “Frag-test_pause”
conditions
4/4/2013: former appendix PNL19-4 (BioNumerics specifications for the E. coli O157
VNTR loci) was moved to SOP PND14 (PulseNet standard operating procedure for
analysis of MLVA data of Shiga toxin-producing Escherichia coli in BioNumerics –
Beckman Coulter CEQ 8000/8800/GeXP data). Former appendices PNL19-5 and PNL19-
6 were renamed PNL19-4 and PNL19-5, respectively.
2/26/14: the three laboratory SOPs for STECO157 (PNL19), and Salmonella serotypes
Typhimurium (PNL21) and Enteritidis (PNL27) using the beckman Coulter
CEQ8000/8800/GeXP platform were combined into a single SOP (PNL19).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 15 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

Appendix PNL19-1
MLVA PCR Primer sequences for STEC O157:H7 and Salmonella serotypes Typhimurium and Enteritidis
Locus Dye1, 2 Forward Primer (5’ to 3’) Reverse Primer (5’ to 3’)
________________________________________________________________________________________________________________________________________________
STECO157:H7
VNTR-3 D2 GG CGG TAA GGA CAA CGG GGT GTT TGA ATT G GAA CAA CCT AAA ACC CGC CTC GCC ATC G
VNTR-34 Quas670 GA CAA GGT TCT GGC GTG TTA CCA ACG G GTT ACA ACT CAC CTG CGA ATT TTT TAA GTC CC
VNTR-9 Quas670 GC GCT GGT TTA GCC ATC GCC TTC TTC C GTG TCA GGT GAG CTA CAG CCC GCT TAC GCT C
VNTR-25 Quas705 GC CGG AGG AGG GTG ATG AGC GGT TAT ATT TAG TG GCG CTG AAA AGA CAT TCT CTG TTT GGT TTA CAC GAC
VNTR-17 D2 GC AGT TGC TCG GTT TTA ACA TTG CAG TGA TGA GGA AAT GGT TTA CAT GAG TTT GAC GAT GGC GAT C
VNTR-19 Quas670 GC AGT GAT CAT TAT TAG CAC CGC TTT CTG GAT GTT C GGG GCA GGG AAT AAG GCC ACC TGT TAA GC
VNTR-36 Quas670 GG CGT CCT TCA TCG GCC TGT CCG TTA AAC GCC GCT GAA AGC CCA CAC CAT GC
VNTR-37 Quas705 GC CGC CCC TTA CAT TAC GCG GAC ATT C GCA GGA GAA CAA CAA AAC AGA CAG TAA TCA GAG CAG C

Salmonella Typhimurium
ST3 Quas705 GT TCT TCT GCA ACG CAG GCA GAT GGC ATG ACG CTG CAA CG
ST5 Quas670 TT TTC GCT CAA CAA ACT T ACA GCA CCA GAA GCA AT
ST7 D2 CG ATT GAC GAT ATC TAT GAC TT GTT TTT CAC GTT TGC CTT TC
ST10 Quas705 CG GGC GCG GCT GGA GTA TTT G GAA GGG GCC GGG CAG AGA CAG C
ST2 Quas670 CA ACG CCT GTT CAG CAA C ATC AAC AGC GGG TGG AT
ST6 D2 AG CAG TGG CTG GCG GGA AAC C GCA GCC GGA CAG GGG ATA AGC C
ST8 Quas705 GC AGG TGT GGC TAT TGG CGT TGA AA GAT GGT GAC GCC GTT GCT GAA GG

Salmonella Enteritidis
SE-1 Quas670 TGT GGG ACT GCT TCA ACC TTT GGG C CCA GCC ATC CAT ACC AAG ACC AAC ACT CTA TGA
SE-2 D2 GTG CTT CCT CAG GTT GCT TTT AGC CTT GTT CG GGG GAA TGG ACG GAG GCG ATA GAC G
SE-8 Quas705 GGT AGC TTG CCG CAT AGC AGC AGA AGT GGC GGC AAG CGA GCG AAT CC
SE-6 Quas670 CTG GTC GCA GGT GTG GC GGT GAC GCC GTT GCT GAA GGT AAT AAC AGA GTC
SE-5 Quas705 GGC TGG CGG GAA ACC ACC ATC GCC GAA CAG CAG GAT CTG TCC ATT AGT CAC TG
SE-3 D2 CGG GAT AAG TGC CAC ATA ACA CAG TCG CTA AGC CGC CAG TGT TAA AGG AAT GAA TGA ACC TGC TGA TG
SE-9 Quas670 CCA CCT CTT TAC GGA TAC TGT CCA CCA GC GGC GTT ACT GGC GGC GTT CG
________________________________________________________________________________________________________________________________________________
1
Only the 5’ of the forward primer is fluorescently labeled
2
Wellred dye used for D2 (black); Quas705 and Quas670 used as more inexpensive replacements for Wellred D3 (green); and D4 (blue). Only the 5’ end of the forward primer is labeled.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 16 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for
data entry and autocalculation
VERSION: REPLACED BY: AUTHORIZED BY:

Page 17 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

VERSION: REPLACED BY: AUTHORIZED BY:

Page 18 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and
autocalculation
VERSION: REPLACED BY: AUTHORIZED BY:

Page 19 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data
entry and autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

Page 20 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and
autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

Page 21 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

Appendix PNL19-4

Instructions to prepare the internal ladders

1. Prepare DNA templates from isolates as described in the protocol step 4.5. Store the templates at -
20oC or -80oC freezer.
1.1 For STEC O157:H7, use the strains EC04PN0139 and EC04PN0570
1.2 For Salmonella serotype Typhimurium, use strains 2009K0825 and 2009K0826
1.3 For Salmonella serotype Enteritidis, use strains H9560 and 2010K0017
2. Use the DNA templates to set up and run the PCR reactions R1 and R2 as described in the protocol
step 4.6.
3. After PCR amplification, pool the R1 and R2 reactions for the two strains into one single PCR tube
to end up with a final volume of 40 µl. Mix by pipetting up and down a few times.
4. A new lot of internal ladder must be tested against the old ladder lot by running them in the same
fragment analysis run.
5. Store the ladder in -20oC or -80oC freezer. It should remain stable at least 5-6 freeze-thaw cycles
for a period of one month.

Expected fragment sizes (bp) of the fifteen fragments (locus VNTR_36 is a null allele in EC04PN0139)
present in the internal ladder as seen in the electropherogram from left to right:
(NOTE: fragment size ranges for the internal ladders are based on multiple independent runs at CDC
and PulseNet Participating Laboratories)

VNTR-25 (Quas705 = D3): 122-124 134-136

VNTR-17 (D2): 149-152 175-178
VNTR-36 (Quas670 = D4): 172-175 NA
VNTR-37 (Quas705 = D3): 194-197 200-203
VNTR-34 (Quas670 = D4): 224-227 260-262
VNTR-19 (Quas670 = D4): 296-299 320-322
VNTR-3 (D2): 392-396 429-431
VNTR-9 (Quas670 = D4): 518-521 566-570

Expected fragment sizes (bp) of the fifteen fragments (locus VNTR_36 is a null allele in EC04PN0139)
present in the internal ladder as listed in the peak file:

VNTR-17 (D2): 149-152 175-178

VNTR-3 (D2): 392-396 429-431
VNTR-25 (Quas705 = D3): 122-124 134-136
VNTR-37 (Quas705 = D3): 194-197 200-203
VNTR-36 (Quas670 = D4): 172-175 NA
VNTR-34 (Quas670 = D4): 224-227 260-262
VNTR-19 (Quas670 = D4): 296-299 320-322
VNTR-9 (Quas670 = D4): 518-521 566-570
VERSION: REPLACED BY: AUTHORIZED BY:

Page 22 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

Expected fragment sizes (bp) of the fourteen fragments present in the internal ladder from the smallest
to the largest in the order they appear in the chromatogram:

ST7 (D2): 130-136 139-145

ST3 (Quas705 = D3): 164-171 174-182
ST5 (Quas670 = D4): 186-188 232-234
ST6 (D2): 247-250 278-280
ST2 (Quas670 = D4): 358-363 387-392
ST10 (Quas705 = D3): 379-381 411-413
ST8 (Quas705 = D3): 580-584 587-592

Expected fragment sizes (bp) of the fourteen fragments present in the internal ladder by the dye in the
order they appear in the peak file:

ST7 (D2): 130-136 139-145

ST6 (D2): 247-250 278-280
ST3 (Quas705 = D3): 164-171 174-182
ST10 (Quas705 = D3): 379-381 411-413
ST8 (Quas705 = D3): 580-584 587-592
ST5 (Quas670 = D4): 186-188 232-234
ST2 (Quas670 = D4): 358-363 387-392

Expected fragment sizes (bp) of the thirteen fragments (SE9 has the same allele in both ladder strains)
present in the internal ladder from the smallest to the largest in the order they appear in the
chromatogram:
SE9 (Quas 670 = D4): 183-186 183-186
SE1 (Quas 670 = D4): 193-196 214-217
SE3 (D2): 197-201 209-214
SE5 (Quas 705 = D3): 201-203 219-221
SE2 (D2): 312-316 353-354
SE8 (Quas 705 = D3): 344-347 433-435
SE6 (Quas 670 = D4): 443-445 476-481

Expected fragment sizes (bp) of the thirteen fragments (SE9 has the same allele in both ladder strains)
present in the internal ladder by the dye in the order they appear in the peak file:

SE3 (D2): 197-201 209-214

SE2 (D2): 312-316 353-354
SE5 (Quas 705 = D3): 201-203 219-221
SE8 (Quas 705 = D3): 344-347 433-435
SE9 (Quas 670 = D4): 183-186 183-186
SE1 (Quas 670 = D4): 193-196 214-217
SE6 (Quas 670 = D4): 443-445 476-481
VERSION: REPLACED BY: AUTHORIZED BY:

Page 23 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

Appendix PNL19-5

Maintenance of the Beckman Coulter CEQ 8000/ 8800/GeXP

After each run

1. Clean the wetting tray thoroughly by first rinsing it with warm tap water and then with
distilled water. Remove any dried gel from the tray and the lid with a paper towel.
Refill the tray with distilled water.
2. The remaining gel can stay in the instrument if it is going to be used within 48 hours.
The shelf-life of the gel at the room temperature is 72 hours.

Instrument is not used for a week or more

1. Refill the wetting tray at least twice a week with distilled water to keep the capillaries
moist at all times.
2. It is recommended to keep a gel in the injection pump at all times to keep the pump
moist. Use leftover gels (< 3.0 ml) that cannot be used for a run anymore.
3. Perform a “Capillary Fill” and a “Manual Purge” (0.1 ml 4 times) once a week to keep
the lines open and the capillaries unblocked.
4. Remove the capillary array if the instrument is not used for more than two weeks to
extend the shelf-life of the array. Replace with an old array and keep performing
capillary fills and manual purges once a week as described above.

Instructions to change the Beckman CEQ capillary array

1. Open the new capillary array package and carefully work loose the plastic covers from
the electrode block, but leave the covers still attached.
2. To release the old capillary array, select the “Release Capillary Array” from the “Run”
pull down menu or click on the capillary array picture in the direct control screen →
the “Remove Capillary Array” dialog box will appear
3. Open the sample access cover
4. Open the capillary access cover
5. To open the capillary temperature control cover, unlatch the two rubber latches
6. To open the manifold access cover and the plenum assembly, loosen the captive
screws
7. Lift the red “eject lever” to release the array fitting
8. Pull out the array fitting with your right hand and the electrode block with your left
hand and set aside
9. Remove the plastic covers from the electrode block and the array fitting of the new
array. Be careful not to break the capillaries!
10. Remove the yellow lens cover from the array fitting. Be careful not to touch the lens!
11. Align the array fitting and the electrode block with the guide pins.
12. Replace the manifold access cover and the plenum assembly. Carefully route the
capillaries through the whole in the plenum assembly.
13. Close the capillary temperature control cover.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 24 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

14. Lower the capillary access cover and the sample access cover to their locking positions
15. Click on “OK” in the “Remove Capillary Array” dialog box → the “Install Capillary
Array” dialog box will appear
16. Change the serial number and click first on “Set to New” and then on “Done” → the
“Confirm Capillary Array Selection” dialog box will appear → select “Yes”
17. Make sure that the instrument has a gel cartridge and a wetting tray in place. Select
“Capillary Fill” from the “Direct Control” drop-down menu. Let the instrument fill the
capillaries (take’s about 3.7 min).
18. Repeat the capillary fill step.
19. Perform three manual purge cycles with 0.1 to 0.4 ml of gel by selecting “Manifold
Purge” from the “Direct Control” drop-down menu or by clicking on the funnel on the
direct control screen.
20. To perform the optical alignment, either select “Optical Alignment” from the “Direct
Control” drop-down menu or click on the lens picture in the direct control screen
21. After the optical alignment has been completed, monitor the fluorescence baseline:
select “Monitor Baseline” from the “Run” drop-down menu → “Monitor Baseline”
dialog box will appear → check the “Enable Monitor Baseline” box and click “OK”
22. To view the baseline trace, switch to data monitor window and view the baseline for
each capillary. Baseline should be less than 5000 units. If it is higher, perform the
following procedure:
a. Release the capillary array and open the sample access cover, the capillary access
cover, the temperature control cover and the manifold access cover
b. Remove the array fitting
c. Carefully wipe clean both sides of the optical window in the array fitting by using a
cotton swab moistened with distilled water. Wipe to one direction only.
d. Dry the optical window using a second swab by wiping to the same direction as
above
e. Clean the manifold access area first by using a moist swab and then with a dry swab
f. Replace the array fitting in the manifold
g. Click on “OK” in the “Remove Capillary Array” dialog box → the “Install
Capillary Array” dialog box will appear
h. Select “Clean Capillaries” button, and click on “Done”
i. Perform two capillary fills and three manual purges
j. Repeat the optical alignment step. If the baseline is still above 5000 units after the
repeated optical alignment, the new capillary array is most likely defective and must
be replaced with another array.
23. To stop the baseline monitoring, go back to the “Monitor Baseline” dialog box and
uncheck the “Enable Monitor Baseline” box and click on “OK”
24. To check the optical scan data, go the Main menu and click on the “Sequencing” icon.
From the “File” drop-down menu, select “Open” → the “Open” dialog box will appear
→ select the “Optical scan data” tab. Find today’s optical scan file by using the filter
(type in today’s date as a start and an end date and click on “Refresh”) → highlight
today’s scan file and click on “OK”. The red and blue peaks in the optical scan data
should start from the galvanometer position 45-50 and end at about 200-210. If the
peaks start much earlier or later, the optical alignment of the instrument is off and may
contribute to increased fragment analysis failure rate. The red and blue peaks should be
superimposed at each galvanometer reading. If they are clearly separated, the optical
VERSION: REPLACED BY: AUTHORIZED BY:

Page 25 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL19
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
Effective Date:
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND 02 26 14
ENTERITIDIS – BECKMAN COULTER CEQ 8000/8800/GeXP PLATFORM

alignment is not optimal. In either case, a service call to the Beckman technical
support is needed.

Instructions to make a new working database

1. Close all open modules.

2. In the main menu of the CEQ software, click on the “Database” icon.
3. From the “File” drop-down menu, select “New Database” → The “New Database”
dialog box will appear
4. Enter a name for the new database by indicating the date on which it was created. For
example: Oct_6_2006.
5. Check the box “Set as a Working Database” and click “OK”.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 26 of 26
 STANDARD OPERATING PROCEDURE FOR ADDING THIOUREA TO 0.5X TBE CODE: PNL20
BUFFER FOR STRAINS OF E.COLI O157:H7, SALMONELLA, VIBRIO, AND OTHER Effective Date:
SPECIES OR GENERA THAT ARE “UNTYPEABLE” BY PFGE 05 18 07

1. PURPOSE: To describe the guidelines for the addition of Thiourea to 0.5X TBE Buffer for strains of E.
coli O157:H7, Salmonella, Vibrio, and other species or genera that are “untypeable” by PFGE.

2. SCOPE: This procedure applies to all PulseNet procedures written in relation to the standardized PFGE
laboratory protocols.

3. DEFINITIONS/TERMS:
3.1 PFGE: Pulsed-field Gel Electrophoresis
3.2 CDC: Centers for Disease Control and Prevention

4. RESPONSIBILITIES/PROCEDURE:
4.1 Make aqueous stock solution containing 10 mg Thiourea/ml (10 μg/μl):
4.1.1 Dissolve 1 gram thiourea in 100 ml sterile Reagent Grade water in clear sterile screw-cap glass
bottle or flask (final concentration is 10 mg/ml). Cover bottle of stock solution with aluminum
foil to protect from light. 1
4.1.2 Safety Caution: Thiourea is a toxic chemical. Weigh thiourea in a chemical fume hood; use
gloves, eye protection, and disposable spatula (e.g., wood tongue depressor) when handling this
chemical. Clean up any spills, and wipe down balance and surrounding area with a moistened
towel. Discard gloves, spatula, weighing paper, etc. as hazardous waste, according to the
guidelines of your institution. Re-cap bottle tightly after use.
4.2 Restrict Salmonella ser. Braenderup H9812 PulseNet standard strain with XbaI and the test samples
with the appropriate restriction enzyme (XbaI, BlnI, NotI, etc.); load restricted plug slices on comb
or in wells of 1% agarose gel. Include a “positive control” on the gel (a slice from a plug that was
“untypeable” without thiourea, but gave a typical PFGE pattern when thiourea was added to the
running buffer).
4.2.1 Note: Do not add thiourea to the melted agarose used for the gel. It will contaminate the gel
form, platform, comb and glassware.
4.3 Make 0.5X TBE with Reagent Grade water; add buffer to electrophoresis chamber and circulate and
cool to 14°C. Put gel in chamber.
4.4 Add the following amount of the Thiourea Stock Solution (final concentration of thiourea is 50 μM)
to the running buffer close to the back of the chamber, near the wells of the gel:
836 μl thiourea stock solution when 2.2 liters of 0.5X TBE is in the chamber and lines
760 μl thiourea stock solution when 2.0 liters of 0.5X TBE is in chamber and lines
4.5 Allow buffer to circulate for 2-3 minutes before starting electrophoresis using PulseNet standard
electrophoresis conditions.
4.6 Stain, de-stain, and document gel according to the appropriate (organism) PulseNet standardized
PFGE protocol.
4.7 When electrophoresis run is over, remove as much of the buffer as possible; pour down sink with tap
water running. Add 2 liters of DI water to the gel chamber and circulate for 15-20 minutes through
the chamber and lines before draining water, adding fresh buffer and using chamber for another gel.

1
Effect of light has not been determined empirically. Do not store solution in a dark bottle because it will not be
possible to tell if solution is discolored or has precipitated.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 2
 STANDARD OPERATING PROCEDURE FOR ADDING THIOUREA TO 0.5X TBE CODE: PNL20
BUFFER FOR STRAINS OF E.COLI O157:H7, SALMONELLA, VIBRIO, AND OTHER Effective Date:
SPECIES OR GENERA THAT ARE “UNTYPEABLE” BY PFGE 05 18 07

4.8 Note: If possible, use the same electrophoresis unit for all thiourea experiments until the long-term
effects (if any) of this chemical on the electrophoresis chamber and tubing have been determined. 2

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

7.1 Efrain Ribot, Ph.D.

CDC
PulseNet Methods Development and Research Unit
(404) 639-3521
[email protected]

8. AMENDMENTS:

2
At CDC, no negative effects have been observed on the gel chambers or tubing after adding thiourea to the 0.5X TBE
running buffer for an average of 2 – 3 gels per month for over five years.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 2
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

1. PURPOSE: to describe the standardized laboratory protocol for molecular subtyping of Shiga
toxin-producing Escherichia coli O157 (STEC O157) and Salmonella enterica serotypes
Typhimurium and Enteritidis.

2. SCOPE: to provide the PulseNet participants with a single protocol for performing MLVA of
STEC O157 and Salmonella serotypes Typhimurium and Enteritidis, thus ensuring inter-laboratory
comparability of the generated results.

3. DEFINITIONS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 VNTR: Variable-number tandem repeat
3.3 DNA: Deoxyribonucleic acid
3.4 DNase: Deoxyribonuclenase
3.5 PCR: Polymerase chain reaction
3.6 HPLC: High purity liquid chromatography
3.7 dNTP: Deoxyribonucleotide triphosphate
3.8 CDC: Centers for Disease Control and Prevention
3.9 SOP: Standard Operating Procedure

4. RESPONSIBILITIES/PROCEDURE
4.1. Biosafety warning: STEC O157 and Salmonella serotypes Typhimurium and Enteritidis
with an infectious dose as low as 100 cells are human pathogens capable of causing
serious disease. Always use a minimum of Biosafety level 2 practices and extreme
caution when transferring and handling strains of these serotypes. Work in a biological
safety cabinet when handling large amounts of cells. Disinfect or dispose of all plastic
ware and glassware that come in contact with the cultures in a safe manner.
4.2. Reagents, supplies and equipment needed for DNA template preparation
4.2.1 Trypticase soy agar with 5 % sheep blood (TSA-SB) or comparable media
4.2.2 1 µl inoculation loops
4.2.3 0.5 ml microcentrifuge tubes
4.2.4 DNase-free, molecular biology -grade water
4.2.5 Vortex
4.2.6 Boiling water bath or thermocycler/thermal block accommodating 0.5 ml tubes
4.2.7 Tabletop centrifuge for high rpm (up to 13,000-14,000 rpm) spinning
4.2.8 Pipets (200 µl) for aliquoting 100 µl of DNase-free, molecular biology-grade water
4.2.9 Filtered Sterile Pipet tips
4.3. Reagents, supplies and equipment needed for PCR
4.3.1 DNA templates from isolates (keep at -20oC or -80ºC freezer for long term)
4.3.2 PCR primers (see appendix PNL23-1)
4.3.2.1 Fluorescent-labeled forward primers
4.3.2.1.1 HPLC-purified
4.3.2.2 Unlabeled reverse primers
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

4.3.2.2.1 Regular gel filtration purification

4.3.2.3 Biosearch Technologies (Novato, CA; www.biosearchtech.com; 1-800-436-
6631) synthesizes primers labeled with the three dyes needed for the protocol
4.3.2.4 Divide the concentrated stocks (100 µM) in portions and store at -80ºC freezer
4.3.2.4.1 One vial should contain enough to prepare 25-50 µl of working solution.
Avoid repetitive freeze-thaw cycles of concentrated primer stocks.
4.3.2.5 The 1.0, 2.5, 5.0, 12.5 and 25.0 µM working solutions can be stored at either -
20oC or -80ºC freezer
4.3.2.6 Prepare new working solutions every month or if a significant drop in the
fluorescence level is observed (for instructions refer to PNQ06_MLVA ABI
certification, appendix PNQ06-5)
4.3.3 96-well polypropylene PCR plates (Fisher, Cat. No. 07-200-613) or Microamp PCR
tubes without caps (Life Technologies, Cat. No. N8010533)
4.3.4 8-well strip caps for the polypropylene plate (Fisher, Cat. No. 07-200-639) or
MicroAmp strip caps for the individual tubes (Life technologies, Cat. No. N8010535)
4.3.5 DNase-free, molecular biology -grade water
4.3.6 1.5 ml Eppendorf microcentrifuge tubes
4.3.7 PCR Nucleotide Mix (ready-to-use dNTP mix containing all four nucleotides; Roche,
Cat. No. 11 814 362 001)
4.3.8 Platinum Taq Polymerase with 50 mM MgCl2 and 10X buffer (Life Technologies, Cat.
No. 10966-034)
4.3.9 PCR Cooling block (VWR International, Cat. No. 62111-762)
4.3.10 DNA Engine (Biorad), GeneAmp (Life Technologies) or similar thermocycler with a
heated lid option and a 96-well block format
4.3.11 Parafilm M, 4” width (VWR, Cat. No. 52858-032)
4.3.12 Complete set (1000 µl, 200 µl, 100 µl, 20 µl, 10 µl and 2 µl) of single channel
pipettors for mastermix set-up (“clean set”)
4.3.13 1-10 µl single channel pipettor for adding DNA templates
4.3.14 Filtered tips for pipettors
4.3.15 Microfuge for low (up to 6,000 rpm) rpm spinning
4.4. Reagents, supplies and equipment needed for Genetic Analyzer 3130
4.4.1 DNase-free, molecular biology -grade water
4.4.2 PCR Cooling block (VWR International, Cat. No. 62111-762)
4.4.3 10 l, 100 l, and 1000 l single channel pipettors
4.4.4 1-10 l and 20-200 µl multichannel pipettors
4.4.5 Filtered pipette tips
4.4.6 Sterile solution basins
4.4.7 1.5 ml Eppendorf microcentrifuge tubes
4.4.8 96-well polypropylene (non-PCR) V-bottom plate (for dilutions; Fisher Scientific, Cat.
No. 07-200-698)
4.4.9 MicroAmp Optical 96-well reaction plates (Life Technologies, Cat. No. 4306737)
4.4.10 96-well plate base (Life Technologies, Cat. No. 4317237)
VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

4.4.11 Rubber septa for 96-well reaction plates (Life Technologies, Cat. No. 4315933)
4.4.12 96-well plate retainer (Life Technologies, Cat. No. 4317241)
4.4.13 Hi-Di Formamide (Life Technologies, 25 ml Cat. No. 4311320)
4.4.14 GeneFlo 625 DNA size standard – ROX, 800 µl (Chimerx, Cat. No. 3125-02)
4.4.15 Multi-Capillary DS-30 (Dye Set D) Matrix Standard Kit (Life Technologies, Cat. No.
4345827)
4.4.15.1 Needed to establish the system dye color spectra for the instrument. Required
when analyzing fragments labeled with FAM, HEX, NED, and ROX.
4.4.15.2 NOTE: DS-33 Matrix Standard (Dye set G5, required for analyzing fragments
labeled with FAM, VIC, NED, PET, and LIZ) is typically installed as default as
part of the instrument installation process
4.4.15.3 In order to install the DS-30 Matrix, follow the instructions of the kit insert and
the “Getting Started Guide”, chapter “Performing a Spectral Calibration”
4.4.16 3130 & 3100 Capillary Array 50 cm (Life Technologies, Cat. No. 4315930)
4.4.17 Genetic Analyzer Buffer (10x) with EDTA, 25 ml (Life Technologies, Cat. No.
402824)
4.4.18 3130 POP7 Performance Optimized Polymer, 3.5 ml (Life Technologies, Cat. No.
4363785)
4.4.19 Parafilm M, 4” width (VWR, Cat. No. 52858-032)
4.4.20 50 ml conical tube
4.4.21 A rinse bottle containing distilled water
4.4.22 Centrifuge with a microtiter plate rotor
4.4.23 Heating block or thermal cycler accommodating a 96-well plate for denaturation
4.4.24 Microfuge for low (up to 6,000 rpm) rpm spinning
4.5. DNA template preparation
4.5.1 Day 0:
4.5.1.1 Streak an isolated colony from test cultures to TSA-SB plate (or comparable
media). Incubate cultures at 37ºC for 14-18 hrs.
4.5.2 Day 1:
4.5.2.1 For each isolate to be typed, aliquot 100 l of sterile, molecular biology-grade
water into 0.5 ml microfuge tubes. Use a sterile, disposable 1 µl loop to pick 2-3
colonies (about half of a loopful); rotate the loop in the microfuge tube to release
the bacteria into the water. Cap and vortex for 10-15 seconds to disperse any
clumps.
4.5.2.2 Place the tubes in a 99-100ºC water bath or heat block for 10-15 minutes. Cool
briefly on ice or in fridge and centrifuge for 10 minutes at 10,000 rpm. Place on
ice or in fridge while preparing PCR reactions. These DNA templates can be
stored at -20oC or -80ºC for several years.
4.6. PCR procedure
4.6.1 Day 1:
4.6.1.1 Fill out, save with the run name, and print an organism specific (copy and paste the
appropriate sizing tables for the controls) MLVA Fragment Analysis ABI
VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

Worksheet (see appendix PNL23-2) with appropriately labeled samples (a

maximum of 46 isolates/plate; 44 unknowns + two positive controls and a
negative control; two wells are reserved for the internal ladder).
4.6.1.1.1 For each isolate, two wells must be labeled as follows: “BNkeyR1” where
“BNkey” represents the isolate-specific state laboratory identification
number (be sure to use the exact same isolate ID that is used in the PFGE
gels uploaded to the national database) and “R1” represents one of the two
specific multiplex PCR reactions (R1, R2).
4.6.1.2 Fill out, and print a PCR mastermix calculation worksheet (see appendices PNL23-
3a, PNL23-3b and PNL23-3c) by typing the number of isolates to be tested (plus
2-3 extra) in the PCR mastermix calculators labeled R1 and R2. This number is
highlighted in RED and is next to “number of samples to be analyzed”. The
mastermixes for reactions 1 and 2 (R1, R2) for one sample are as follows:

STEC O157:H7 (appendix PNL23-3a)

R1 Volume ( µl) Final conc.

PCR water 5.30
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
VNTR-3F (25 µM) 0.27 0.67 µM
VNTR-3R (25 µM) 0.27 0.67 µM
VNTR-34F (5 µM) 0.24 0.12 µM
VNTR-34R (5 µM) 0.24 0.12 µM
VNTR-9F (5 µM) 0.24 0.12 µM
VNTR-9R (5 µM) 0.24 0.12 µM
VNTR-25F (2.5 µM) 0.20 0.05 µM
VNTR-25R (2.5 µM) 0.20 0.05 µM
Taq (5 U/µl) 0.20 1U
= 9.00

R2 Volume (µl) Final conc.

PCR water 5.92
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
VNTR-17F (5 µM) 0.30 0.15 µM
VNTR-17R (5 µM) 0.30 0.15 µM
VNTR-19F (1 µM) 0.16 0.016 µM
VNTR-19R (1 µM) 0.16 0.016 µM
VNTR-36F (1 µM) 0.11 0.011 µM
VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

VNTR-36R (1 µM) 0.11 0.011 µM

VNTR-37F (2.5 µM) 0.07 0.017µM
VNTR-37R (2.5 µM) 0.07 0.017µM
Taq (5 U/µl) 0.20 1U
= 9.00

Salmonella serotype Typhimurium (appendix PNL23-3b)

R1 Volume ( µl) Final conc.

PCR water 3.81
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.45 2.25 mM
dNTPs (10 mM) 0.20 0.20 mM
ST3-F (5 µM) 0.10 0.05 µM
ST3-R (5 µM) 0.10 0.05 µM
ST5-F (25 µM) 0.60 1.50 µM
ST5-R (25 µM) 0.60 1.50 µM
ST7-F (5 µM) 0.80 0.40 µM
ST7-R (5 µM) 0.80 0.40 µM
ST10-F (2.5 µM) 0.12 0.03 µM
ST10-R (2.5 µM) 0.12 0.03 µM
Taq (5 U/µl) 0.30 1.50 U
= 9.00

R2 Volume (µl) Final conc.

PCR water 4.38
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.32 1.60 mM
dNTPs (10 mM) 0.20 0.20 mM
ST2-F (25 µM) 0.36 0.90 µM
ST2-R (25 µM) 0.36 0.90 µM
ST6-F4 (5 µM) 0.56 0.28 µM
ST6-R2 (5 µM) 0.56 0.28 µM
ST8-F3 (5 µM) 0.48 0.24 µM
ST8-R2 (5 µM) 0.48 0.24 µM
Taq (5 U/µl) 0.30 1.50 U
= 9.00

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

Salmonella serotype Enteritidis (appendix PNL23-3c)

R1 Volume (µl) Final conc.

PCR water 4.91
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
SE1-F (2.5 µM) 0.20 0.05 µM
SE1-R (2.5 µM) 0.20 0.05 µM
SE2-F (12.5 µM) 0.32 0.40 µM
SE2-R (12.5 µM) 0.32 0.40 µM
SE8-F (2.5 µM) 0.28 0.07 µM
SE8-R (2.5 µM) 0.28 0.07 µM
SE6-F (12.5 µM) 0.34 0.43 µM
SE6-R (12.5 µM) 0.34 0.43 µM
Taq (5 U/µl) 0.20 1.00 U
= 9.00

R2 Volume (µl) Final conc.

PCR water 5.52
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
SE5-F (2.5 µM) 0.20 0.05 µM
SE5-R (2.5 µM) 0.20 0.05 µM
SE3-F (12.5 µM) 0.40 0.50 µM
SE3-R (12.5 µM) 0.40 0.50 µM
SE9-F (2.5 µM) 0.08 0.02 µM
SE9-R (2.5 µM) 0.08 0.02 µM
Taq (5 U/µl) 0.20 1.00 U
= 9.00

4.6.1.2.1. NOTE: these primer concentrations serve as a starting point. Since

laboratory-specific factors, such as the age of the primer stocks, calibration
status of the thermocyclers and pipettes, etc. affect amplification efficiency,
each laboratory will have to re-optimize the primer concentrations for
optimal detection of all targets. However, any other parameters stated in the
SOP should not be changed.
4.6.1.3 Thaw all reagents and supplies needed for PCR reactions and place on ice; keep
primers light protected as much as possible

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

4.6.1.3.1 NOTE: PCR mastermixes should be set up in a clean hood that is dedicated
just for this purpose and where no cultures or DNA are handled.
4.6.1.4 Prepare the two separate PCR mastermixes in 1.5 ml Eppendorf tubes following
the instructions in the PCR mastermix calculation worksheet (see appendices
PNL23-3a, PNL23-3b and PNL23-3c). Keep the mastermix on ice while
preparing. Add the mastermix components in the following order: water, 10x
PCR buffer, Mg2Cl, dNTPs, primers, and then finally Taq polymerase. Mix the
reaction mixture by vortexing briefly.
4.6.1.4.1 NOTE: All components except Taq polymerase should be vortexed
thoroughly before adding to the mastermix. Taq may be briefly centrifuged
with low rpm, if necessary, to pull the enzyme down to the bottom of the
tube.
4.6.1.5 Place a 96-well PCR plate or required number of PCR tubes in a PCR cooling
block.
4.6.1.6 Dispense 9.0 l of each mastermix into the appropriate rows of the 96-well
polypropylene plate / PCR tubes as noted in the PCR template worksheet (see
appendix PNL23-2).
4.6.1.7 Add 1 l of PCR water to each of the two different wells representing the
negative controls of the two reactions.
4.6.1.8 Add 1.0 l of DNA template to each of the two different wells representing the
two PCR reactions for each isolate to be tested
4.6.1.9 Add the positive controls (it is recommended to run the positive control in
duplicate).
4.6.1.9.1 Use STEC O157 strain EDL933 (ATCC 43895) as a positive control. The
internal ladder to be used will be comprised of pooled PCR products of the
isolates EC04PN0139 and EC04PN0570 (see appendix PNL23-4 for
instructions for ladder preparation).
4.6.1.9.2 Use S. enterica serotype Typhimurium strain LT2 (ATCC 29946) as a
positive control. The internal ladder to be used will be comprised of pooled
PCR products of the isolates CDC_2009K0825 and CDC_2009K0826 (see
appendix PNL23-4 for instructions for ladder preparation).
4.6.1.9.3 Use S. enterica serotype Enteritidis strain K1891 (ATCC 25928) as a positive
control. The internal ladder to be used will be comprised of pooled PCR
products of the isolates H9560 and 2010K0017 (see appendix PNL23-4 for
instructions for ladder preparation).
4.6.1.10 Cover all wells / tubes with 8-well strip caps and firmly clamp down to avoid any
evaporation during PCR amplification.
4.6.1.10.1 Recommendation: briefly spin down the plate / tubes to remove any air
bubbles.
4.6.1.11 Program and save the following two PCR cycling conditions:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

STEC O157:H7 and Salmonella serotype Enteritidis

“O157-SEMLVA”
* 95ºC for 5 min Step 1
* 94ºC for 20 sec Step 2
* 65ºC for 20 sec Step 3
* 72ºC for 20 sec Step 4
* Go to step 2, 34x Step 5
* 72ºC for 5 min Step 6
* Indefinite hold at 4ºC Step 7

Salmonella serotype Typhimurium

“STMLVA”
* 95ºC for 5 min Step 1
* 94ºC for 20 sec Step 2
* 63ºC for 20 sec Step 3
* 72ºC for 20 sec Step 4
* Go to step 2, 34x Step 5
* 72ºC for 5 min Step 6
* Indefinite hold at 4ºC Step 7

4.6.1.12.1 NOTE: Make sure to use the heated lid option on the PCR block and tube
(calculated) temperature control.
4.6.1.13 When the PCR is complete store the amplification products light-protected at 4ºC
until ready to run on the sequencer. If the fragment analysis is not performed the
same day, the plate should be stored at -20oC or -80ºC. The PCR products are
stable for approximately one month, when stored frozen.
4.7. Initial setup of Genetic Analyzer 3130 instrument:
4.7.1 NOTE: steps 4.7.3 and 4.7.4 only need to be performed before the very first run
4.7.2 Click on the “Run 3130 Data Collection v 3.0” icon. The “Service Console” window
will appear. If the connections are functioning properly, the “Messaging Service”,
“Data Service”, “Instrument Service” and “Viewer” icons will change from red circles
to green squares. The main window of the “Foundation Data Collection” software will
open. Check to make sure system status is green.
4.7.3 Set up a results group:
4.7.3.1 On the left side of the window under “GA Instruments”, highlight “Results group”
and click on “New”.
4.7.3.2 The “Results Group Editor” window will appear.
4.7.3.3 Under “General” tab, name the new results group as “MLVA1”.
4.7.3.3.1 When the results group reaches its upper limit set up a new results group with
a different name (MLVA2, MLVA3…).
VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

4.7.3.4 Under “Analysis” tab, select “GeneMapper-Generic” from the “Analysis type”
drop-down menu. Leave “Analysis actions” unchecked.
4.7.3.5 Under “Destination” tab, keep the default root destination
(E:\AppliedBiosystems\udc\DataCollection\Data).
4.7.3.6 Under “Naming” Tab, select “Sample Name” from the first “Sample File Name
Format” drop-down menu, and select “Plate Name” from the first “Run Folder
Name Format” drop-down menu. Leave all other fields blank.
4.7.3.7 Under “Automated Processing” tab, leave the default “Only when the results group
is complete” checked.
4.7.4 Set up running conditions:
4.7.4.1 Under “GA Instruments”, click on + to expand “ga3130” subfolders.
4.7.4.2 Highlight “Protocol Manager”. The instrument protocols will be listed on the right
side of the window.
4.7.4.3 Click on “New”, and the “Protocol Editor” window will appear.
4.7.4.3.1 Name the new protocol “FragTest”.
4.7.4.3.2 Leave the protocol type as “Regular”.
4.7.4.3.3 From the “Run Module” drop-down menu, select “Fragment Analysis
50_POP7_1” as running conditions.
4.7.4.3.3.1 NOTE: These are the instrument default running conditions for 50
cm capillary array and POP7 polymer. You can check the running
conditions and modify them by highlighting “Module Manager”
subfolder under ga3130 and by double clicking on the protocol
name. The default conditions are:
* Oven_Temperature: 60oC
* Poly Fill Vol: 7300 Steps
* Current Stability: 5.0 μAmps
* PreRun_Voltage: 15.0 kVolts
* Pre-Run-Time: 180 sec.
* Injection_Voltage: 1.6 kVolts
* Injection_Time: 15 sec.
* Voltage_Number_Of_Steps: 30 nk
* Voltage_Step_Interval: 15 sec.
* Data_Delay_Time: 200 sec.
* Run_Voltage: 15.0 kVolts
* Run time: 1800 sec.
4.7.4.3.4 From the “Dye Set” drop-down menu, select “D” as dye set.
4.8. Genetic Analyzer 3130 instrument preparation before each run
4.8.1. Day 1
4.8.1.1 Make sure the service console is open and all components are green. Make sure a
capillary array is installed in the instrument. For installation, follow the
instructions of the “Install Capillary Wizard”. You can find the wizards by

VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

expanding the 3130 subfolder and by highlighting “Manual Control”. The

“Wizards” drop-down menu will appear on top of the window.
4.8.1.2 Set up the plate run on the 3130 manually, as detailed below, or follow the steps in
the appendix PNL23-5 to create a template that can be used to import the plate set
up from a separate Excel file.
4.8.1.2.1 Under the ga3130, highlight “Plate Manager”.
4.8.1.2.2 Click on “New” and a “New Plate Dialog Window” will appear.
4.8.1.2.3 Name the run following the standardized PulseNet naming system: use the
unique identifier code that was assigned to your laboratory by PulseNet for
the first two to four letters of the file name. The next two spaces will indicate
the year and the next four spaces will indicate the month and the date the run
was performed. For example GA070426 is a run made at the GA Public
Health Laboratory on April 26th 2007. If several runs are performed the same
day, separate the file names by using sequential numbers, for example
GA070426-1, GA070426-2.
4.8.1.2.4 From the “Application” drop-down menu, select “GeneMapper-Generic”
4.8.1.2.5 Type in the Owner Name and the Operator Name.
4.8.1.2.6 Click “OK” and the “GeneMapper Plate Editor” window will appear.
4.8.1.2.6.1 Type in the sample IDs.
4.8.1.2.6.2 Select “MLVA1” from the “Results Group 1” drop-down menu for the
first sample, highlight the results group column, and select “Fill
Down” from the “Edit” drop-down menu.
4.8.1.2.6.3 Select “FragTest” from the “Instrument Protocol 1” drop-down menu,
highlight the instrument protocol column, and select “Fill Down”
from the “Edit” drop-down menu.
4.8.1.2.6.4 Click “OK”.
4.8.1.3 Install the POP7 polymer in the instrument
4.8.1.3.1 Expand the 3130 subfolder and highlight “Manual Control”. The “Wizards”
drop-down menu will appear on top of the window.
4.8.1.3.2 Follow the instructions of the “Replenish Polymer Wizard”
4.8.1.3.2.1 NOTE1: if an old polymer (been on the instrument > 7 days) is
switched to a new one follow the instructions of the “Water Wash
Wizard” until you reach the step in which the array port should be
flushed. At this point, if you don’t see an air bubble in the port,
cancel out the water wash wizard and perform a spatial calibration
with a capillary fill. If you see an air bubble in the port perform the
flush as instructed by the wizard and then continue to capillary fill
as instructed.
4.8.1.3.2.2 NOTE2: Use distilled water that has been heated to 37-40oC for water
wash.
4.8.1.3.2.3 NOTE3: Water wash should be performed once a week.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

4.9. Fragment analysis sample preparation

4.9.1 Day 1
4.9.1.1 NOTE: The fragment analysis method is not organism specific therefore; STEC
O157 and Salmonella serotypes Typhiumurium and Entertidis may be run on a
single fragment analysis plate.
4.9.1.2 Thaw the Hi-Di Formamide, the GeneFlo 625 DNA size standard and the internal
ladder (see appendix PNL23-4) and place on ice.
4.9.1.2.1 NOTE: aliquot Hi-Di Formamide (500 µl / tube) and the size standard (50
µl / tube) in order to avoid frequent freeze-thaw cycles.
4.9.1.3 Prepare a 96-well V-bottom plate for diluting the PCR reactions. Using a 200 µl
multichannel pipettor and a solution basin, dispense 19 µl of molecular-grade
water in the required number of wells.
4.9.1.4 Remove the plate / tubes with the PCR reactions from the thermocycler. Briefly
spin down the plate / tubes, if necessary. Use a 10 µl multichannel pipettor to
transfer 1 µl of each PCR reaction directly across to the corresponding set of
wells in the dilution plate. In order to avoid cross-contamination, remove the strip
cap from just one column at a time and recap the column before opening the next
one.
4.9.1.5 For the internal ladder, combine R1 and R2 PCR products from the four PCR
reactions of both internal ladder isolates into one tube to end with a total of 40 µl.
Mix well by pipetting up and down a few times and add 3 l of internal ladder in
two wells.
4.9.1.6 Using a 200 µl multichannel pipettor, mix the dilutions by pipetting up and down
a few times. Cover the plate with parafilm and put in the fridge or on ice.
4.9.1.7 Prepare a fragment analysis master mix containing DNA size standard and Hi-Di
Formamide for the samples following the calculations indicated in the table
below. The fragment analysis mastermix calculations can also be performed
using the autocalculate box at the bottom of the MLVA Fragment Analysis ABI
Worksheet (see appendix PNL23-2). Vortex briefly and place on ice.

Reagents Frag. anal. mastermix

Hi-Di Formamide 8 µl x (# samples +3)=
GeneFlo 625 bp size standard 1 µl x (# samples +3)=

4.9.1.8 Place a MicroAmp Optical 96-well sample plate in a cold block. Aliquot 9 µl of
the prepared fragment analysis mastermix to the required number of wells. Cover
the plate loosely with Parafilm.
4.9.1.9 Using the 10 µl multichannel pipettor, add 1 µl of 1:20 diluted PCR reactions to
the appropriate columns in the sample plate. Keep sliding the Parafilm sheet from
column to column to keep track of the sample order.
4.9.1.10 Denaturate templates by heating the reaction plate uncovered at 95oC for 3 min

VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

4.9.1.11 Manually turn on the oven of the 3130, by using the Command options under the
Manual Control submenu to change the set point to 60oC.
4.9.1.12 While the templates denaturate, prepare 30 ml 1X running buffer.
4.9.1.12.1 Add 3 ml of 10X Genetic Analyzer buffer in a 50 ml conical tube.
4.9.1.12.2 Add purified water to bring the total volume up to 30 ml.
4.9.1.12.3 Mix well.
4.9.1.13 Briefly spin down the sample plate to remove any air bubbles.
4.9.1.14 Seal the plate with the rubber septa and place the sample plate in a plate base.
Snap the plate retainer onto the plate and the plate base.
4.9.1.15 Place the plate assembly and the buffer into the Genetic Analyzer 3130.
4.9.1.15.1 Push on the Tray button at the front of the Genetic Analyzer 3130 to
bring the autosampler to the forward position. Open the instrument doors.
4.9.1.15.2 Place the plate assembly on the autosampler in position A or B with the
notched end of the plate base away from you.
4.9.1.15.3 Add 1X running buffer to the anode and cathode reservoirs.
4.9.1.15.4 Add distilled water to the waste and rinse reservoirs.
4.9.1.15.5 Close the instrument doors and wait for the green light to illuminate.
4.9.1.16 Start the run after making sure the system status is green.
4.9.1.16.1 Expand 3130 computer name icon.
4.9.1.16.2 Highlight “Run Scheduler”.
4.9.1.16.3 Click on “Find All”.
4.9.1.16.4 Highlight the plate name for the run.
4.9.1.16.5 Link the plate by clicking on the yellow squares on the right side of the
window that correspond to the position of the plate.
4.9.1.16.6 The “Start Run” button will turn green indicating that the run can be started.
Click on this button, and then click “OK” on the alert window. The run will
start.
4.10. Viewing and exporting data from the Genetic Analyzer 3130
4.10.1 Day 2
4.10.1.1 NOTE: Steps 4.10.1.3 and 4.10.1.4 only need to be performed before the very
first analysis.
4.10.1.2 Double-click on the shortcut icon for GeneMapper v.4.0 and enter the appropriate
password to access the software. The main menu window will open.
4.10.1.3 Set up the size standard:
4.10.1.3.1 From the “Tools” drop-down menu, select “GeneMapper Manager”. A
“GeneMapper Manager” window will open.
4.10.1.3.2 Select the “Size Standards” tab.
4.10.1.3.3 Click on “New”, leave the default option “Basic or Advanced” checked and
click “OK”. The “Size Standard Editor” window will appear.
4.10.1.3.4 Name the new size standard “GeneFlo 625”.
4.10.1.3.5 Leave the default option “Red” as “Size Standard Dye”.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

4.10.1.3.6 Enter sizes for each peak in the table (refer to the GeneFlo 625 product
insert for the peak sizes).
4.10.1.3.7 When finished, click “OK”.
4.10.1.4 Set up analysis method:
4.10.1.4.1 From the “Tools” drop-down menu, select “GeneMapper Manager”. A
“GeneMapper Manager” window will open.
4.10.1.4.2 Select the “Analysis Methods” tab.
4.10.1.4.3 Click on “New”, leave the default option “Microsatellite” checked and click
“OK”. The “Analysis Methods Editor” window will appear.
4.10.1.4.4 Name the new method “PNMLVA” and click “OK”.
4.10.1.4.5 Highlight the new method name “PNMLVA” and click “Open”.
4.10.1.4.6 Select the “Peak Detector” tab and change “Peak Detection Algorithm” to
“Advanced” from the drop-down menu.
4.10.1.4.7 Input the following analysis settings and click “OK” when finished:
* Analysis: Full Range
* Sizing: Partial Sizing
* Start Size: 50
* Stop Size 625
* Smoothing: none
* Baseline window: 51 pts
* Size Calling Method: Local Southern Method
* Peak Amplitude Thresholds
* B: 600
* G: 600
* Y: 600
* R: 20
* O: 50
* Min. Peak Half Width: 2 pts
* Polynomial Degree: 2
* Peak Window Size: 21 pts
* Slope Threshold
* Peak Start: 0.0
* Peak End: 0.0
4.10.1.5 From the “File” drop-down menu select “Add Samples to Project”.
4.10.1.6 Find the folder containing the data file to be analyzed: My Computer → E: →
Applied Biosystems → UDC → Data Collection → Data.
4.10.1.7 Highlight the desired file(s) and click on “Add to List”. File(s) will appear in the
window on the right. Click “Add” below the file list to return to the original
screen.
4.10.1.8 Samples in the selected file(s) will be listed in a new window and the “Analyze”
(play) button appears in green color in the toolbar indicating that the files are
ready to be analyzed.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

4.10.1.9 Select the size standard GeneFlo 625 for the first sample, highlight the size
standard column, and select “Fill Down” from the “Edit” drop-down menu.
4.10.1.10 Select the analysis method PNMLVA for the first sample, highlight the analysis
method column, and select “Fill Down” from the “Edit” drop-down menu.
4.10.1.11 Click on the “Play” icon.
4.10.1.12 Name the project with the run name (for example, “GA070426”) and click “OK”
4.10.1.13 A successful analysis is indicated by green squares. Yellow triangles indicate
problematic components (i.e. missing size standard peaks). Red circles indicate
that results fell below acceptable quality values. Samples with yellow or red
circles in the SQ column should be selected for re-analysis.
4.10.1.13.1 To resolve failed analyses due to sub-optimal molecular marker peak
profile (i.e. miscalling of peaks), select a row with a yellow triangle or red
circle in the SQ column and click on the “Size Match Editor” icon on the
toolbar. The “Size Match Editor” view will appear.
4.10.1.13.2 Place the cursor near the X-axis to activate the magnifying lens, and
then pull up (mouse left-click and hold) to zoom in a specific area to
facilitate editing
4.10.1.13.3 Left-click at the base of a peak to select. Right-click and select “Add’,
“Delete”, or “Change”.
4.10.1.13.4 Select the correct molecular weight for the selected peak from the drop-
down menu. Repeat this process for all other miscalled peaks. Click “OK”
when finished.
4.10.1.13.5 After the size standard has been adjusted, click the “Play” button to re-
analyze the data. After a successful analysis, the samples will have green
squares under the SQ column. If the size standard cannot be adjusted, the
reaction is considered a fragment analysis failure and must be re-run
4.10.1.14 Check the fragment result data (the fluorescent peaks) for each well by
highlighting the well ID and by clicking on the “Display Plots” icon on the
toolbar.
4.10.1.14.1 Make sure that all VNTRs amplified in the positive control and that the
fragment sizes are within the range specified in the appendix PNL23-2 and
record the fragment sizes on the MLVA Fragment Analysis ABI
Worksheet.
4.10.1.14.2 The size calling for the internal ladder should also be within the range
specified in the appendix PNL23-2 (or PNL23-4).
4.10.1.14.3 Write down any failed reactions in the MLVA Fragment Analysis ABI
Worksheet. Make a note of non-specific bands and primer-dimers.
4.10.1.15 Export the peak file:
4.10.1.15.1 NOTE: the following columns should appear in the exported table in the
following order from left to right: “Dye/Sample Peak”, “Sample File
Name”, “Marker”, “Size”, “Height”, “Area”, “Data Point”. You can modify
the format of the table by selecting “Table setting editor” from the “Tools”
VERSION: REPLACED BY: AUTHORIZED BY:

Page 14 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

drop-down menu. Select the “Genotype” tab and make sure that the boxes
for the above mentioned columns are checked and no additional boxes are
checked.
4.10.1.15.2 Highlight the samples for which you want to export peak data.
4.10.1.15.3 Click on the “Display Plots” icon on the toolbar.
4.10.1.15.4 Click on the “Sizing Table” icon on the toolbar and a table will appear
below the electropherograms.
4.10.1.15.5 From the “File” drop-down menu, select “Export Table”.
4.10.1.15.6 Select the location (for example a flash drive) where you want to export
the data.
4.10.1.15.7 Name the export file with the run name (for example GA070426) and
make sure the file type is a tab-delimited text (.txt) file.
4.10.1.16 The remaining gel can stay in the instrument if it is going to be used within 7
days.
4.10.1.16.1 NOTE: To extend life of polymer, remove after run, place in
refrigerator, and replace with an old polymer (on instrument longer than 7
days) or water bottle until next use. Polymer should not be on instrument
for more than a total of 7 days.

5. FLOW CHART:

6. REFERENCES:
6.1 Hyytiä-Trees, E., Smole, S. C., Fields, P. I.., Swaminathan, B., and Ribot, E. M. (2006)
Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-
number tandem repeat analysis (MLVA) of Shiga toxin-producing Escherichia coli O157
(STEC O157). Foodborne Pathog. Dis. 3, 118-131.
6.2 Hyytia-Trees, E., Lafon, P., Vauterin, P., and Ribot, E. (2010) Multi-laboratory validation
study of standardized multiple-locus VNTR analysis (MLVA) protocol for Shiga toxin-
producing Escherichia coli O157 (STEC O157): a novel approach to normalize fragment
size data between capillary electrophoresis platforms. Foodborne Path. Dis. 7, 129-136.

7. CONTACTS:
7.1 Eija Trees, D.V.M., Ph.D.
PulseNet Next Generation Subtyping Methods Unit, CDC
(404) 639-3672
[email protected]
7.2 Patti Lafon
PulseNet Next Generation Subtyping Methods Unit, CDC
(404) 639-2828
[email protected]
7.3 Ashley Sabol
PulseNet Next Generation Subtyping Methods Unit, CDC
VERSION: REPLACED BY: AUTHORIZED BY:

Page 15 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

(404) 639-2947
[email protected]

8. AMENDMENTS:
7/19/2011: appendix PNL23-5 was added. This document gives instructions on how to export a
plate set up from the plate manager in order to create a template that can be used to import plate set
ups from a separate Excel file.
4/10/2013: instructions to perform the water wash at step 4.8.1.3.2.1 were changed to reflect the fact
that performing the array port flush after loosening up the ferule, as instructed by the instrument
water wash wizard, may damage the array when performed multiple times.
4/10/2013: instructions to prepare the 1X running buffer were changed in step 4.9.1.11. The buffer
should be prepared in a conical tube instead of a graduated cylinder to minimize possible
contamination and ensure adequate mixing.
4/10/2013: former appendix PNL23-4 (BioNumerics specifications for the E. coli O157 VNTR loci)
was moved to SOP PND16 (PulseNet standard operating procedure for analysis of MLVA data of
Shiga toxin-producing Escherichia coli in BioNumerics – Applied Biosystems Genetic Analyzer
3130/3500 data). Former appendices PNL23-5 and PNL23-6 were renamed PNL23-4 and PNL23-5,
respectively.
2/26/2014: the three laboratory SOPs for STECO157 (PNL23), and Salmonella serotypes
Typhimurium (PNL24) and Enteritidis (PNL26) using the ABI 3130 platform were combined into a
single SOP (PNL23).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 16 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

Appendix PNL23-1
MLVA PCR Primer sequences for STEC O157:H7 and Salmonella serotypes Typhimurium and Enteritidis
Locus Dye1 Forward Primer (5’ to 3’) Reverse Primer (5’ to 3’)
_____________________________________________________________________________________________________________________________ ___________________
STEC O157:H7
VNTR-3 CalRed590 GG CGG TAA GGA CAA CGG GGT GTT TGA ATT G GAA CAA CCT AAA ACC CGC CTC GCC ATC G
VNTR-34 FAM GA CAA GGT TCT GGC GTG TTA CCA ACG G GTT ACA ACT CAC CTG CGA ATT TTT TAA GTC CC
VNTR-9 FAM GC GCT GGT TTA GCC ATC GCC TTC TTC C GTG TCA GGT GAG CTA CAG CCC GCT TAC GCT C
VNTR-25 HEX GC CGG AGG AGG GTG ATG AGC GGT TAT ATT TAG TG GCG CTG AAA AGA CAT TCT CTG TTT GGT TTA CAC GAC
VNTR-17 CalRed590 GC AGT TGC TCG GTT TTA ACA TTG CAG TGA TGA GGA AAT GGT TTA CAT GAG TTT GAC GAT GGC GAT C
VNTR-19 FAM GC AGT GAT CAT TAT TAG CAC CGC TTT CTG GAT GTT C GGG GCA GGG AAT AAG GCC ACC TGT TAA GC
VNTR-36 FAM GG CGT CCT TCA TCG GCC TGT CCG TTA AAC GCC GCT GAA AGC CCA CAC CAT GC
VNTR-37 HEX GC CGC CCC TTA CAT TAC GCG GAC ATT C GCA GGA GAA CAA CAA AAC AGA CAG TAA TCA GAG CAG C

Salmonella Typhimurium
ST3 HEX GT TCT TCT GCA ACG CAG GCA GAT GGC ATG ACG CTG CAA CG
ST5 FAM TT TTC GCT CAA CAA ACT T ACA GCA CCA GAA GCA AT
ST7 CalRed590 CG ATT GAC GAT ATC TAT GAC TT GTT TTT CAC GTT TGC CTT TC
ST10 HEX CG GGC GCG GCT GGA GTA TTT G GAA GGG GCC GGG CAG AGA CAG C
ST2 FAM CA ACG CCT GTT CAG CAA C ATC AAC AGC GGG TGG AT
ST6 CalRed590 AG CAG TGG CTG GCG GGA AAC C GCA GCC GGA CAG GGG ATA AGC C
ST8 HEX GC AGG TGT GGC TAT TGG CGT TGA AA GAT GGT GAC GCC GTT GCT GAA GG

Salmonella Enteritidis
SE-1 FAM TGT GGG ACT GCT TCA ACC TTT GGG C CCA GCC ATC CAT ACC AAG ACC AAC ACT CTA TGA
SE-2 CallRed590 GTG CTT CCT CAG GTT GCT TTT AGC CTT GTT CG GGG GAA TGG ACG GAG GCG ATA GAC G
SE-8 HEX GGT AGC TTG CCG CAT AGC AGC AGA AGT GGC GGC AAG CGA GCG AAT CC
SE-6 FAM CTG GTC GCA GGT GTG GC GGT GAC GCC GTT GCT GAA GGT AAT AAC AGA GTC
SE-5 HEX GGC TGG CGG GAA ACC ACC ATC GCC GAA CAG CAG GAT CTG TCC ATT AGT CAC TG
SE-3 CallRed590 CGG GAT AAG TGC CAC ATA ACA CAG TCG CTA AGC CGC CAG TGT TAA AGG AAT GAA TGA ACC TGC TGA TG
SE-9 FAM CCA CCT CTT TAC GGA TAC TGT CCA CCA GC GGC GTT ACT GGC GGC GTT CG
________________________________________________________________________________________________________________________________________________
1
Only the 5’ of the forward primer is fluorescently labeled

VERSION: REPLACED BY: AUTHORIZED BY:

Page 17 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

Page 18 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

VERSION: REPLACED BY: AUTHORIZED BY:

Page 19 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

Page 20 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and
autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

Page 21 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM
Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

Page 22 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

Appendix PNL23-4

Instructions to prepare the internal ladders

1. Prepare DNA templates from isolates as described in the protocol step 4.5. Store the templates at -20oC or -
80oC freezer.
1.1 For STEC O157:H7, use the strains EC04PN0139 and EC04PN0570
1.2 For Salmonella serotype Typhimurium, use strains 2009K0825 and 2009K0826
1.3 For Salmonella serotype Enteritidis, use strains H9560 and 2010K0017
2. Use the DNA templates to set up and run the PCR reactions R1 and R2 as described in the protocol step 4.6.
3. After PCR amplification, pool the R1 and R2 reactions for the two strains into one single PCR tube to end
up with a final volume of 40 µl. Mix by pipetting up and down a few times.
4. A new lot of internal ladder must be tested against the old ladder lot by running them in the same fragment
analysis run.
5. Store the ladder in -20oC or -80oC freezer. It should remain stable at least 5-6 freeze-thaw cycles for a
period of one month.

Expected fragment sizes (bp) of the fifteen fragments (locus VNTR_36 is a null allele in EC04PN0139)
present in the STEC O157:H7 internal ladder as listed in the peak file:
VNTR-36 (B): 170 - 172 NA
VNTR-34 (B): 222 - 224 260 - 262
VNTR-19 (B): 296 - 298 320 - 322
VNTR-9 (B): 518 - 522 565 - 569
VNTR-25 (G): 127 - 128 138 - 140
VNTR-37 (G): 192 - 194 198 - 200
VNTR-17 (Y): 153 - 155 177 - 179
VNTR-3 (Y): 397 - 401 433 - 435

Expected fragment sizes (bp) of the fifteen fragments (locus VNTR_36 is a null allele in EC04PN0139)
present in the STEC O157:H7 internal ladder as they appear in the electropherogram:
VNTR-25 (G): 127 - 128 138 - 140
VNTR-17 (Y): 153 - 155 177 - 179
VNTR-36 (B): 170 - 172 NA
VNTR-37 (G): 192 - 194 198 - 200
VNTR-34 (B): 222 - 224 260 - 262
VNTR-19 (B): 296 - 298 320 - 322
VNTR-3 (Y): 397 - 401 433 - 435
VNTR-9 (B): 518 - 522 565 - 569

VERSION: REPLACED BY: AUTHORIZED BY:

Page 23 of 26
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

Expected fragment sizes (bp) of the fourteen fragments present in the Salmonella serotype Typhimurium
internal ladder as listed in the peak file:
ST5 (B): 184 - 185 231 - 233
ST2 (B): 370 - 373 399 - 401
ST3 (G): 177 - 180 188 - 192
ST10 (G): 383 - 384 413 - 415
ST8 (G): 582 - 584 589 - 591
ST7 (Y): 137 - 139 147 - 151
ST6 (Y): 253 - 255 283 - 285

Expected fragment sizes (bp) of the fourteen fragment present in the Salmonella serotype Typhimurium
internal ladder as they appear in the electropherogram:
ST7 (Y): 137 - 139 147 - 151
ST3 (G): 177 - 180 188 - 192
ST5 (B): 184 - 185 231 - 233
ST6 (Y): 253 - 255 283 - 285
ST2 (B): 370 - 373 399 - 401
ST10 (G): 383 - 384 413 - 415
ST8 (G): 582 - 584 589 - 591

Expected fragment sizes (bp) of the thirteen fragments (SE9 has the same allele in both ladder strains)
present in the Salmonella serotype Enteritidis internal ladder as listed in the peak file:
SE9 (B) 181 - 184 181 - 184
SE6 (B): 446 - 447 479 - 482
SE1 (B) 190 - 193 211 - 213
SE5 (G): 201 - 203 218 - 221
SE8 (G): 346 - 350 433 - 436
SE3 (Y): 199 – 203 211 - 215
SE2 (Y): 317.5 - 324 363 - 364

Expected fragment sizes (bp) of the fifteen fragments (SE9 has the same allele in both ladder strains)
present in the Salmonella serotype Enteritidis internal ladder as they appear in the electropherogram:
SE9 (B) 181 - 184 181 – 184
SE1 (B) 190 - 193 211 - 213
SE3 (Y): 199 - 203 211 - 215
SE5 (G): 201 - 203 218 - 221
SE2 (Y): 317.5 - 324 363 - 364
SE8 (G): 346 - 350 433 - 436
SE6 (B): 446 - 447 479 - 482

NOTE: fragment size ranges for the internal ladders are based on multiple independent runs at CDC and
PulseNet Participating Laboratories
VERSION: REPLACED BY: AUTHORIZED BY:

Page 24 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

Appendix PNL23-5

Steps for exporting and importing plate set ups on the ABI 3130

Note: the procedure described below will require the sequencer computer to have Microsoft Office. If the
sequencer computer does not have Microsoft Office, the exported file needs to be saved on a flash drive so that
steps 8 through 15 can be performed on a computer with Microsoft Office.

1. Under “GA Instruments”, click on the + to expand the “ga3130” subfolders on the left-hand menu
2. Highlight “Plate Manager” and on the right side of the window click “Find All” to list all of the available
plates
3. Find the most recent full plate. Click on the desired plate and then click the “Export...” button at the bottom
of the screen
3.1. NOTE: If you do not have a full plate, create a new one by filling in each sample ID space (A01
through H12) with “test” and saving it with the plate name “MLVA_Template”. Be sure to add the
results group “MLVA1” and instrument protocol “FragTest” for each sample. It is necessary to have all
required wells filled in when exporting so that they will be available in your exported file
4. In the export window, create a new folder on the desktop by clicking the “Desktop” icon on the left side of
the window. Find the “Create New Folder” button on the top right side of the window and name the new
folder “MLVA Plate Setup”
5. Save the file to this folder by using your new plate name (e.g. - CDC101020) and clicking “Save”
6. A window will pop up letting you know that the plate has been successfully exported. Click “OK”
7. Minimize the 3130 Viewer and open the “MLVA Plate Setup” folder on the desktop
8. Right-click your plate name, select “Open With” and in the submenu select Microsoft Office Excel.
9. Sample IDs can be typed or copied and pasted from a separate Excel file into the spaces next to the correct
wells under the “Sample Name” heading. The first sample ID must be directly under the “Sample Name”
heading
9.1. NOTE: Do not change/delete any of the column headings. The fields must be in the same format when
importing as they were when exported. Additionally, the software will not import IDs with special
characters (e.g. - ! , / , ) , etc) or spaces. You can use underscores and dashes
10. The “Container Name” should be changed to match the plate/file name, and initials should be placed under
the “Owner” and ”Operator” headings
10.1. NOTE: If the container name is not changed it cannot be imported because it will be recognized as a
plate that is already in the system
11. Once all of the IDs are inserted, make sure that for each sample ID and all controls the priority is “100”, the
results group name “MLVA1” is under the “Results Group 1” heading, and the running method “FragTest”
is under the “Instrument Protocol 1” heading
12. For all unused wells, everything under the headings “Well”, “Priority”, “Results Group 1”, and “Instrument
Protocol 1” must be deleted. This can be done by clicking the first cell to be deleted under these columns
and dragging until the entire section is highlighted. Right-click in the highlighted area and from the drop-
down menu select “Clear Contents”
13. Under the “File” drop-down menu select “Save”
VERSION: REPLACED BY: AUTHORIZED BY:

Page 25 of 26
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL23
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3130 02 26 14
PLATFORM

14. A warning window will appear asking if the workbook should be saved in the “Text (tab-delimited)” format.
Click “Yes”
15. Exit Microsoft Excel, clicking “No” when prompted to save changes
16. Go back to the ABI 3130 plate manager window and click the “Import...” button at the bottom of the screen
17. In the import window, navigate to the recently created plate under Desktop/MLVA Plate Setup, select the
appropriate .txt file and click “Open”
18. When the file has been imported, a “Progress” window will appear stating that the plate was “successfully
imported”. Click “OK”
18.1. NOTE: For repeated use, plate templates can be created by exporting a plate to the “MLVA Plate
Setup” folder. Save the file as a tab-delimited text file with ‘test’ as the sample ID and with a new plate
name (e.g. “MLVA_Template”). After you have filled out the information for a new run on the template,
save it in step 13 with the standardized run name (name (e.g. CDC101020).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 26 of 26
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

1. PURPOSE: To describe standardized protocols for molecular subtyping of botulinum toxin producing
clostridia by Pulsed-field Gel Electrophoresis (PFGE).

2. SCOPE: To provide the PulseNet participants with the same procedures for performing PFGE of
botulinum toxin producing clostridia thus ensuring interlaboratory comparability of the generated results.

3. DEFINITIONS/TERMS:

3.1 PFGE: Pulsed-field Gel Electrophoresis

3.2 DNA: Deoxyribonucleic acid
3.3 CDC: Centers for Disease Control and Prevention
3.4 CLRW: Clinical Laboratory Reagent Water

4. RESPONSIBILITIES/PROCEDURE:

BIOSAFETY WARNING:

All samples received must be considered infectious. Botulinum toxin producing clostridia and/or botulinum toxin may
be present in a variety of food products, clinical materials (serum, feces) and environmental samples (soil, surface water).
Exposure to botulinum toxin is the primary laboratory hazard. The toxin may be absorbed after ingestion or following
contact with the broken skin, eyes, or mucous membranes, including the respiratory tract. Accidental parenteral
inoculation may also represent a significant exposure to toxin. Broth cultures grown under optimal conditions for toxin
production may contain 2,000,000 mouse LD50 per ml of toxin.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all
activities with materials known to or that may potentially contain botulinum toxin. Personal protective equipment (PPE)
i.e., gloves, lab coat, safety glasses and/or face shield should be worn at all times when handling anything that has come
into contact with the organism or toxin including pipette tips and plastic transfer pipettes used to transfer liquids. All
liquids require disinfection using a freshly prepared 10% bleach solution. Solutions of sodium hypochlorite (0.1%) or
sodium hydroxide (0.1N) readily inactivate the toxin and are recommended for decontaminating work surfaces and spills
of cultures or toxin.

Please read all instructions carefully before starting protocol. All plasticware, glassware, pipets, spatulas, etc. that come in
contact with the cell suspensions or plugs should be disinfected with 10% bleach for at least 1 hour before they are washed and
reused.

SELECT AGENTS REQUIREMENTS:

All PFGE materials, including restricted plugs, may contain viable bacteria until the start of electrophoresis. Botulinum
toxin producing clostridia are Select Agents (SA), and according to the APHIS/CDC SA Regulations all parts of the
following procedure until electrophoresis must be performed by SA approved personnel and within SA approved space.
In addition, long term stored plugs are subject to SA inventory requirements, as defined by APHIS/CDC SA Regulations.
Please refer to www.selectagents.gov for additional information.

PREPARATION OF PFGE PLUGS FROM TEST CULTURES

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

Day 1
1. Streak each test culture for colony isolation onto Anaerobic Blood Agar Plate – CDC formulation (ANA-BAP).

2. Incubate the plates in an anaerobic chamber overnight at 35°C ± 2°C.

Day 2
1. Label small tubes (12-mm x 75-mm Falcon tubes or equivalent) with culture numbers.

2. Prepare Cell Suspension Buffer (100 mM Tris:100 mM EDTA, pH 8.0) as follows:

200 ml of 1 M Tris-HCl, pH 8.0

400 ml of 0.5 M EDTA, pH 8.0
Dilute to 2000 ml with sterile Ultrapure water (Clinical Laboratory Reagent Water (CLRW))

Note: Acceptable options for CLRW are type I or Milli-Q water.

3. Transfer ≈1.5 ml of Cell Suspension Buffer (CSB) to the labeled tubes. Use a sterile polyester-fiber or cotton swab
that has been moistened with sterile CSB to remove some of the growth from the ANA-BAP plate; suspend cells in
CSB by spinning the swab gently so cells will be evenly dispersed and formation of aerosols is minimized.

4. Adjust concentration of cell suspensions to a milky turbidity of ~1 McFarland (0.18-0.20 on Dade Microscan
Turbidity Meter) by diluting with sterile CSB or by adding additional cells.

5. Pipette 1000 µl of the cell suspensions into sterile microcentrifuge tubes and spin for 5 minutes at 5,000 rpm.

6. Remove the supernatants, resuspend cells in 1000 µl of the cell suspension buffer and spin for 5 minutes at 5,000
rpm. Decant supernatants in waste container containing bleach.

7. Remove the supernatants and resuspend cells in 500 µl of the cell suspension buffer. Decant supernatants in
waste container containing bleach.

8. Use these washed cells to inoculate two EYA plates per sample for confluent growth (250 µl per plate), using the
Kirby –Bauer technique.

9. Incubate the plates for 18-72 hours in an anaerobic chamber at 35°C ± 2°C.

Note: Remove plates from incubation as soon as there is sufficient growth for testing. 18 hours is
typically optimal.

Day 3
1. Turn on shaker water bath or incubator (55°C ± 2°C ) and stationary water baths (55°C ± 2°C and 37°C ± 2°C ).

2. Add bottles of sterile Ultrapure water (CLRW) and TE buffer to the 55°C water bath to warm for the washing
procedural steps.

Note: An acceptable option for CLRW is type I or Milli-Q water.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

3. Prepare 2X Cell Lysis Buffer (12mM Tris, 2M NaCl, 200 mM EDTA, 1% Brij 58, 0.4%
Deoxycholate, 5% Sarcosyl) as follows:

1.2 ml of 1 M Tris-HCl, pH 8.0

40 ml of 5 M NaCl
40 ml of 0.5 M EDTA, pH 8.0
1 g Brij 58
0.4 g Deoxycholate
5 g Sarkosyl
Dilute to 100 ml with sterile Ultrapure water (CLRW)

Note: An acceptable option for CLRW is type I or Milli-Q water.

4. Prepare Lysozyme (Sigma L7651 or equivalent) stock solution (20 mg/mL in TE) as follows:
a. Weigh out 100 mg Lysozyme (keep the container of Lysozyme on ice)
b. Add 5 mL TE buffer, swirl to mix
c. Aliquot 250 uL amounts into small eppendorf tubes and freeze for future use.

5. Prepare Mutanolysin (Sigma M9901 or equivalent) stock solution (5 U/μL in TE) as follows:
a. Add 1 mL TE buffer to vial of lyophilized Mutanolysin, swirl to mix
b. Aliquot 50 μL amounts into small eppendorf tubes and freeze for future use.

6. Take out tubes of Lysozyme (20 mg/ml) and Mutanolysin (5U/µl) needed from the
-20ºC freezer and pre-warm 2X Cell Lysis Buffer to 55 ºC ± 2°C.

7. Prepare 1.2% SeaKem Gold agarose in TE Buffer (10 mM Tris:1 mM EDTA,

pH 8.0) for PFGE plugs as follows:

a. Weigh 0.12 g (or 0.24 g) SeaKem Gold (SKG) agarose into 250 ml screw-cap flask
b. Add 10.0 ml (or 20.0 ml) TE Buffer; swirl gently to disperse agarose.
c. Loosen or remove cap and cover loosely with clear film, and microwave for 30-sec;
mix gently and repeat for 10-sec intervals until agarose is completely dissolved.
d. Recap flask and place in 55°C ± 2°C water bath and equilibrate the agarose in the
water bath for 15 minutes or until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: SeaKem Gold agarose works well for making PFGE plugs because it provides added strength to
the plugs minimizing breakage of plugs during the lysis and washing steps. The time and
temperature needed to completely dissolve the agarose is dependent on the specifications of the
microwave used, and will have to be determined empirically in each laboratory.

8. Label small tubes (12-mm x 75-mm Falcon tubes or equivalent) with culture numbers.
9. Prepare PIV Buffer as follows:

5 ml of 1 M Tris-HCl, pH 8.0
100 ml of 5 M NaCl
Dilute to 500 ml with sterile Ultrapure water (CLRW)

Note: An acceptable option for CLRW is type I or Milli-Q water.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

10. Transfer ≈1.5 ml of PIV Buffer to the labeled tubes. Use a sterile polyester-fiber or cotton swab that has been
moistened with sterile PIV to remove some of the growth from the agar plate; suspend cells in PIV by spinning swab
gently so cells will be evenly dispersed and formation of aerosols is minimized.

Note: If a large number of samples are being prepared, it is recommended that they are prepared in batches of ~10 samples at
a time.

11. Adjust concentrations of cell suspensions to 0.68-0.72 as measured on the Dade Microscan Turbidity Meter by diluting
with sterile PIV Buffer or by adding additional cells. These cell suspension concentrations give satisfactory results at
CDC; each laboratory may need to establish the optimal concentration needed for satisfactory results.

Note: Cell suspensions need to be at room temperature when concentration is checked.

12. Pipette 1000 µl of the cell suspensions into microcentrifuge tubes and spin for 5 minutes at
5,000rpm.

13. Calculate the volume of 2X cell lysis buffer and Proteinase K required per sample as follows:

Note: Stock concentrations of Proteinase K may vary by lot and will need to be calculated for
each shipment. Adjust total volume to compensate by changing amount of 2X cell lysis buffer
used. The final concentration of Proteinase K for plugs is 0.665 mg/ml.

Example Calculation:
(stock conc. of Proteinase K) (X) = (final conc. desired) (final volume)

(stock conc. of Proteinase K) (X) = (0.665 mg/ml) (1 ml)

(0.665 mg/ml) x (1 ml) = volume of Proteinase K needed (ml)

(stock conc. of Proteinase K)

Total volume of 2X cell lysis buffer + Proteinase K = 316 μl

316 μl – volume of Proteinase K = volume of 2X cell lysis buffer needed (μl)

14. Remove the supernatants and resuspend cells in the calculated volume of 2X cell lysis buffer that has been pre-
warmed to 55ºC ± 2°C. Then add 80 µl of lysozyme (20 mg/ml) and incubate in the waterbath for 20 minutes at
55°C ± 2°C. Decant supernatants in waste container containing bleach.

15. After removing the samples from the waterbath, add 4 µl of mutanolysin (5U/µl) and the calculated volume of
proteinase K, and incubate another 10 minutes at 37ºC ± 2°C.

CASTING PLUGS

Label wells of PFGE plug molds with culture number. When reusable plug molds are used, put strip of tape on lower part of
reusable plug mold before labeling wells.

Note: Unused plug agarose can be kept at room temperature and reused 1-2 times. Microwave on low-
medium power for 10 -15 sec and mix; repeat for 5 -10 sec intervals until agarose is completely
melted. This agarose melts rapidly!

1. Add 400 μl (0.4 ml) melted 1.2% SeaKem Gold agarose to the cell suspensions; mix by gently pipetting mixture up
and down a few times. Maintain temperature of melted agarose by keeping flask in beaker of warm water (55-60ºC).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

2. Immediately, dispense part of mixture into appropriate well(s) of reusable plug mold. Do not
allow bubbles to form. Two plugs of each sample can be made from these amounts of cell
suspension and agarose. Allow plugs to solidify at room temperature for 10-15 min.

Note: If plugs are being prepared from a large number of samples, it is recommended that they are prepared in batches of ~10
samples at a time.

LYSIS OF CELLS IN AGAROSE PLUGS

Note: Two plugs of the same strain can be lysed in the same 50-ml tube.

1. Label 50-ml polypropylene screw-cap tubes with culture numbers.

2. Prepare ES Buffer as follows:

495 ml of 0.5 M EDTA, pH 8.0
5 ml of 10 % Sarcosyl

3. Add 5 ml of ES Buffer to each labeled 50 ml tube.

4. Calculate the volume of Proteinase K needed per sample as follows:

Note: Stock concentrations of Proteinase K may vary by lot and will need to be calculated for each shipment.
The final concentration of Proteinase K per tube is 0.14 mg/ml.

Calculation:
(stock conc. of Proteinase K) (X) = (final conc. desired) (final volume)

(stock conc. of Proteinase K) (X) = (0.14 mg/ml) (5 ml)

(0.14 mg/ml) x (5 ml) = volume of Proteinase K needed (ml)

(stock conc. of Proteinase K)

Add the calculated volume of Proteinase K to each labeled 50 ml tube containing ES Buffer.

5. Trim excess agarose from top of plugs with scalpel, razor blade or similar instrument. Open reusable plug
mold and transfer plugs from mold with a 6-mm wide spatula to appropriately labeled tubes containing ES
Buffer + Proteinase K. Be sure plugs are under buffer and not on side of tube.

Note: Ensure that the green screen caps are in place on conical tubes containing the plugs to prevent
the loss or damage of the plug

6. Place both sections of the plug mold, spatulas, and scalpel in 10% bleach. Soak them for 1 hour before
washing them.

7. Place tubes in a rack and incubate in the 55°C ±2°C shaker water bath or incubator for a minimum of 2 h (4 h is
optimal) with constant agitation (40-70 rpm). Be sure water level is above level of lysis buffer in tubes if using
a water bath.

WASHING OF AGAROSE PLUGS AFTER CELL LYSIS

Note: Most laboratories will find that their plugs are sufficiently stable to perform the following washing steps
at 54-55ºC. However, if you notice that your plugs are nicked along the edges or breaking it will be necessary for
your laboratory to lower the water bath or incubator to 50ºC for the following washing steps.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 23
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

1. Remove tubes from water bath and carefully pour off ES Buffer into an appropriate discard container; plugs can
be held in tubes with the green screen caps.

Note: It is important to remove all of the liquid during this and subsequent wash steps by touching edge
of tube or screened cap on an absorbent paper towel.

2. Add 20 ml sterile Ultrapure water (CLRW) that has been preheated to 55ºC ± 2°C to each tube and shake the
tubes in a 55°C ± 2°C water bath for 15 min at 70 RPM. Decant used distilled water into a waste container
containing bleach.

Note: An acceptable option for CLRW is type I or Milli-Q water.

3. Pour off water from the plugs and repeat wash step with 20 ml of preheated water (Step 2) one more time.

4. Pour off water, add a minimum of 20 ml preheated (55ºC ± 2°C) sterile TE Buffer, and shake the tubes in the
55ºC ± 2°C water bath for 15 min at 70 RPM. Decant used TE into a waste container containing bleach.

5. Pour off TE and repeat wash step with preheated TE five more times.

6. Decant last wash and add 15 ml sterile TE. Continue with step 1 in "Restriction Digestion" section or store
plugs in TE Buffer at 4ºC until needed. Plugs can be transferred to smaller tubes containing 1 ml of TE for
storage.

Day 4

RESTRICTION DIGESTION OF DNA IN AGAROSE PLUGS

Note: A small slice of the plug or the entire plug (made in disposable plug molds) can be digested with the
restriction enzyme. Restriction digestion of a small slice of the plug is recommended because less enzyme is required
and other slices of the plug can be subjected to restriction analysis with other enzymes. Restriction analysis with a
secondary enzyme is important in situations where the PFGE patterns obtained with the primary enzyme from two or
more isolates are indistinguishable.

1. Label 1.5 ml microcentrifuge tubes with sample numbers; label 3 (10-well gel) or 4 (15-well gel) tubes for
Salmonella ser. Braenderup H9812 standards.

2. Prepare 1X restriction buffer by diluting the appropriate 10X restriction buffer (provided with each restriction
enzyme by the vendor 1:10 with sterile Ultrapure water (CLRW) according to the following tables:

Note: An acceptable option for CLRW is type I or Milli-Q water.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 23
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

SmaI digestion buffer

Reagent μl/Plug Slice

Sterile Ultrapure Water 180 μl

Restriction Buffer 4 20 μl

Total Volume 200 μl

XbaI digestion buffer

Reagent μl/Plug Slice

Sterile Ultrapure Water 180 μl

Restriction Buffer H 20 μl

Total Volume 200 μl

XhoI digestion buffer

Reagent μl/Plug Slice

Sterile Ultrapure Water 178 μl

Restriction Buffer 4 20 μl
100X BSA 2 μl

Total Volume 200 μl

Note: Use buffer H (Roche) for the standard plugs and buffer 4 (New England Biolabs) for the sample plugs.
Buffer 4 requires the addition of BSA when the plugs are going to be digested with XhoI.

3. Add 200 μl diluted restriction buffer (1X) to the corresponding (samples or standards) 1.5 ml
microcentrifuge tubes.

4. Carefully remove plug from TE with spatula and place in a sterile disposable Petri dish or on
a large glass slide.

5. Cut a 2.0 to 2.5 mm wide slice from test sample plugs with a single edge razor blade
(or scalpel, coverslip, etc.) and transfer to tube containing appropriate diluted restriction buffer. Be sure
plug slice is under buffer. Replace rest of plug into the original tube that contains TE buffer. Store at 4ºC.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 23
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

Note: The shape and size of the plug slice that is cut will depend on the size of the comb teeth that are used for
casting the gel. PulseNet recommends that the combs with larger teeth (10 mm wide teeth) be used to cast the gels
because computer analysis of the gel lanes is more accurate and less tedious than analysis of gel lanes cast with
combs with the smaller teeth (5.5-mm). The number of slices that can be cut from the plugs will depend on the skill
and experience of the operator, integrity of the plug, and whether the slices are cut vertically or horizontally (plugs
made in disposable molds).

6. Cut three or four 2.0 to 2.5 mm wide slices from plug of the S. ser. Braenderup H9812 standard and
transfer to tubes with diluted restriction buffer H. Be sure plug slices are under buffer. Replace rest of
plug in original tube that contains TE buffer. Store at 4ºC.

7. Incubate sample and standard plug slices in 37ºC water bath (for plugs that will be digested with
XbaI or XhoI) or 25ºC water bath (for plugs that will be digested with SmaI) for 10 minutes.

8. Prepare a restriction enzyme master mix with the appropriate restriction enzyme per sample according
to the following tables. Prepare enough for each plug slice plus one additional aliquot.

SmaI (20U/μl stock concentration)

Reagent μl/Plug Slice

Sterile Ultrapure Water 177.5 μl

10X Restriction Buffer 4 20 μl

Enzyme (50 U/sample) 2.5 μl

Total Volume 200 μl

Incubate 25oC, 4 hours

XbaI (20U/μl stock concentration)

Reagent μl/Plug Slice

Sterile Ultrapure Water 175 μl

10X Restriction Buffer H 20 μl

Enzyme (100 U/sample) 5 μl

Total Volume 200 μl

Incubate 37oC, 2 hours

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 23
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

XhoI (20U/μl stock concentration)

Reagent μl/Plug Slice

Sterile Ultrapure Water 173 μl

10X Restriction Buffer 4 20 μl

100X BSA 2 μl
Enzyme (100 U/sample) 5 μl

Total Volume 200 μl

Incubate 37oC, 3 hours

Note: Digest Salmonella standard plugs with Roche XbaI and sample plugs with New England Biolabs SmaI or
XhoI. These restriction enzymes give satisfactory results at CDC. Restriction enzymes provided by other vendors
have not been evaluated. Be certain that the restriction buffer that is used is recommended by the vendor for the
corresponding restriction enzyme and calculate the volume of enzyme needed to achieve the same final
concentrations as indicated in the tables. Keep vials of restriction enzymes on ice or in insulated storage box (-20˚C)
at all times.

a. Addition of Bovine Serum Albumin (BSA): Several restriction enzyme vendors specifically recommend
the addition of 1X BSA to enzyme restriction mixtures. However, BSA can be added to all enzyme
restriction mixtures and may assist in reducing the incidence of incomplete restriction. If BSA is added
to the enzyme reaction mixture, the volume of BSA added should be deducted from the volume of water
to maintain the total volume of 200 μl per slice.

9. After incubation, remove buffer from plug slice using a pipet fitted with 200-250 μl tip all the
way to bottom of tube and aspirate buffer. Be careful not to damage the plug slice with pipet tip
and that plug slice is not discarded with pipet tip.

10. Add 200µl of the appropriate restriction enzyme cocktail to each of the tubes, making sure the
plug is completely submerged in the solution. Close the tubes and mix by gently tapping.

11. Incubate the plug slices with restriction enzymes at each of their respective temperatures and
times.

LOADING PFGE PLUG SLICES AND POURING AGAROSE GEL

1. Confirm that water bath is equilibrated to 55°C ±2°C.

2. Make volume of 0.5X Tris-Borate EDTA Buffer (TBE) that is needed for both the gel and electrophoresis
running buffer according to one of the following tables.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

5X TBE:
Reagent Volume in milliliters (ml)
5X TBE 200 210 220 230 240 250
1
Reagent Grade Water 1800 1890 1980 2070 2160 2250

Total Volume of 0.5X TBE 2000 2100 2200 2300 2400 2500

10X TBE:
Reagent Volume in milliliters (ml)
10X TBE 100 105 110 115 120 125
Reagent Grade Water 1900 1995 2090 2185 2280 2375

Total Volume of 0.5X TBE 2000 2100 2200 2300 2400 2500

2. Make 1% SeaKem Gold (SKG) agarose in 0.5X TBE as follows:

a. Weigh appropriate amount of SKG into 500 ml screw-cap flask.
b. Add appropriate amount of 0.5X TBE; swirl gently to disperse agarose.
c. Loosen or remove cap and cover loosely with clear film, and microwave for 60-sec; mix gently and
repeat for 15-sec intervals until agarose is completely dissolved.
d. Recap flask and return to 55°C ±2°C water bath and equilibrate the agarose in the water bath for 15
minutes or until ready to use.

Mix 1.0 g agarose with 100 ml 0.5X TBE for 14-cm-wide gel form (10 wells)
Mix 1.5 g agarose with 150 ml 0.5X TBE for 21-cm-wide gel form (15 wells)

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

4. Place the gel mold on a leveling table and adjust until perfectly leveled. Place the comb holder so the front part
(side with small metal screws) and teeth face the bottom of gel frame and the comb teeth touch the gel platform.

5. Remove restricted plug slices from the water baths. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE.
Incubate at room temperature for 5 min.

6. Remove plug slices from tubes; put comb on bench top and load plug slices on the bottom of the comb teeth as
follows:

a. Load S. ser. Braenderup H9812 standards on teeth (lanes) 1, 5, 10 (10-well gel) or on

teeth 1, 5, 10, 15 (15-well gel).
b. Load samples on remaining teeth.

7. Remove excess buffer with tissue. Allow plug slices to air dry on the comb for a minimum of 15 minutes.

1
De-ionized water (does not need to be sterilized).
VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

8. Position comb in gel mold and confirm that the plugs slices are correctly aligned on the bottom of the comb
teeth, that the lower edge of the plug slice is flush against the black platform, and there are no bubbles.

9. Carefully pour the agarose (cooled to 55°C ± 2°C) into the gel mold and allow the gel to solidify for a
minimum of 15 minutes at room temperature.

10. Put black gel frame in electrophoresis chamber and add 2.2 L freshly prepared 0.5X TBE. Close cover of unit.
(The amount of buffer needed depends on whether residual buffer was left in tubing or if unit was flushed with
water after the last gel was run.)

11. Turn on cooling module (14ºC), power supply, and pump (set at ≈70 to achieve a flow rate of
1 liter/minute).

12. After the gel has solidified, add 5-10 ml of 0.5X TBE to the comb and gently remove from the gel.

13. Unscrew and remove end gates from gel mold; remove excess agarose from sides and bottom of
casting platform with a tissue. Keep gel on casting platform and carefully place gel inside black
gel frame in electrophoresis chamber.

14. Add 860 µl of Thiourea (10mg/ml) to 0.5X TBE in the electrophoresis chamber. Close cover of chamber.

SAFETY WARNING: Thiourea is a toxic chemical. Weigh thiourea in a chemical fume hood; use gloves, eye
protection, and disposable spatula when handling this chemical. Clean up any spills, and wipe down balance and
surrounding area with a moistened towel. Discard gloves, spatula, weighing paper, etc. as hazardous waste,
according to the guidelines of your institution. Re-cap bottle tightly after use.

ELECTROPHORESIS CONDITIONS

1. Select the following conditions on the CHEF MAPPER:

a. Select the following conditions on the CHEF MAPPER

Auto Algorithm
30 kb - low MW
600 kb - high MW
Initial switch time: 0.5 s
Final switch time: 40 s
Change run time to 18 - 20 h (See note below)
(Default values: Initial switch time = 2.16 s; Final switch time = 54.17 s)

b. Select following conditions on CHEF-DR III

Initial switch time: 2.2 s
Final switch time: 54.2 s
Voltage: 6 V
Included Angle: 120°
Run time: 18-20 h (See note below)

Note: The electrophoresis running times recommended above are based on the equipment and reagents
used at the CDC. Run times may be different in your laboratory and will have to be optimized for
your gels so that the lowest band in the S. ser. Braenderup H9812 standard migrates 1.0 - 1.5 cm

VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

from the bottom of the gel. Make note of the initial milliamp (mAmp) reading on the instrument.
The initial mAmps should be between 110-170 mAmps. A reading outside of this range may
indicate that the 0.5X TBE buffer was prepared improperly and the buffer should be remade.

Day 5

STAINING AND DOCUMENTATION OF PFGE AGAROSE GEL

1. When electrophoresis run is over, turn off equipment; remove and stain gel with ethidium bromide. Dilute 50 μl
of ethidium bromide stock solution (10 mg/ml) with 500 ml of reagent grade water (this volume is for a staining
box that is approximately 14-cm x 24-cm; a larger container may require a larger amount of staining solution).
Stain gel for 20 minutes in covered container with gentile agitation.

Note: Use extreme caution when handling Ethidium Bromide, as it is carcinogenic and a mutagenic
chemical. Stock solutions of 10 mg/ml Ethidium Bromide (EtBr) in water are available from
several commercial companies (Amresco X328; Bio-Rad, 161-0433; Sigma, E-1510). CDC
does not recommend disposing of EtBr down the drain. Aqueous solutions containing EtBr can
be filtered through charcoal or degraded using activated carbon destaining or “tea” bags from
Amresco (E732-25 Destaining Bags) or other companies, which effectively and safely remove
EtBr from solutions and gels. Once the EtBr is removed, the treated aqueous solutions can be
discarded down the drain. If you have further questions about EtBr please refer to the
Material Safety Data Sheets (MSDS) provided by the vendor or manufacturer.

2. Destain gel in approximately 500 ml reagent grade water for 60 min; changing water every 20 minutes. Capture
image using a Gel Doc 1000, 2000, EQ, or XR, or equivalent documentation system. If too much background is
observed destain for an additional 30-60 min.

a. Alternative Method for PFGE Gel Staining (GelStar): Dilute 40 ul of GelStar (10,000X stock solution) into
400 ml of 1X TBE. Stain gel for 60 minutes in covered container with gentile agitation.

Note: Destaining is not necessary when staining the gel with GelStar. After staining, proceed to capturing the
image with a gel documentation system. Stock solutions of GelStar are available from Lonza, 50535. Use the
same precautions when handling and disposing of GelStar as indicated above for EtBr.

3. Capture image using a Gel Doc 1000, 2000, EQ, or XR, or equivalent documentation system.

4. Follow directions given with the imaging equipment to save gel image as an *.img or *.1sc file; convert this
file to *.tif file for analysis with the BioNumerics software program. The gel image should fill the entire
window of the imaging equipment (computer) screen (without cutting off wells or lower bands). Ensure
that the image is in focus and that there is little to no staturation (over-exposure) in the bands. Additional
instructions are provided in PNL07 of the PulseNet QA/QC manual.

5. Drain buffer from electrophoresis chamber and discard. Rinse chamber with 2-4 L reagent grade
water or, if unit is not going to be used for several days, flush lines with water by letting pump
run for 5-10 min before draining water from chamber and hoses. Remove and clean loose pieces
of agarose in the electrophoresis chamber.

6. If the lowest band in the H9812 standard does not migrate within 1 -1.5 cm of the bottom of the
gel, the run time will need to be determined empirically for the conditions in each laboratory.

Please note the following if PFGE results do not have to be available within 24-28 hours:
VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

1. Plugs can be lysed for longer periods of time (3-16 hours).

2. The washing steps with TE to remove the lysis buffer from the PFGE plugs can be done for longer periods of
time (30-45 min) and at lower temperatures (37°C or room temperature). They can be started on Day 1 and
finished on Day 2 after overnight refrigeration of the plugs in TE.

3. The restriction digestion can be done for longer periods of time (3-16 hours).

Use of trade names and commercial sources is for identification purposes only and does not imply endorsement by CDC
or the U.S. Department of Health and Human Services.

ALTERNATIVE PROCEDURE FOR PFGE OF CLOSTRIDIUM BOTULINUM

Some strains may not be typeable by the method described above. The following procedure can be used as an alternative
method to perform PFGE on botulinum toxin producing clostridia.

PREPARATION OF PFGE PLUGS FROM TEST CULTURES

Day 1

1. Streak each test culture for colony isolation onto Egg Yolk Agar Plate.

2. Incubate the plates at 35± 2°C under anaerobic conditions until isolated colonies are present (18-48 hours).

Day 2

1. Inoculate a single colony from each plate into 10ml of Trypticase Peptone Glucose Yeast Extract (TPGY)
medium.

2. Incubate the TPGY tubes at 35± 2°C under anaerobic conditions until growth is evident in the TPGY media.

Day 3

1. Prepare 2X Cell Lysis Buffer (12mM Tris, 2M NaCl, 200 mM EDTA, 1% Brij 58, 0.4%, Deoxycholate, 5%
Sarcosyl) as follows:

1.2 ml of 1 M Tris-HCl, pH 8.0

40 ml of 5 M NaCl
40 ml of 0.5 M EDTA, pH 8.0
1 g Brij 58
0.4 g Deoxycholate
5 g Sarkosyl
Dilute to 100 ml with sterile Ultrapure water (CLRW)

Note: Acceptable options for CLRW is type I or Milli-Q water.

2. Prepare PIV Buffer as follows:

5 ml of 1 M Tris-HCl, pH 8.0
VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

100 ml of 5 M NaCl
Dilute to 500 ml with sterile Ultrapure water (CLRW)

Note: Acceptable options for CLRW is type I or Milli-Q water.

3. Prepare Lysozyme (Sigma L7651 or equivalent) stock solution (20 mg/mL in TE) as follows:
a. Weigh out 100 mg Lysozyme (keep the container of Lysozyme on ice)
b. Add 5 mL TE buffer, swirl to mix
c. Aliquot 250 uL amounts into small eppendorf tubes and freeze for future use.

4. Prepare Mutanolysin (Sigma M9901 or equivalent) stock solution (5 U/μL in TE) as follows:
a. Add 1 mL TE buffer to vial of lyophilized Mutanolysin, swirl to mix
b. Aliquot 50 μL amounts into small eppendorf tubes and freeze for future use.

5. Prepare 1.8% SKG Gold Agarose in TE Buffer (10 mM Tris: 1 mM EDTA, pH 8.0) for PFGE plugs as follows:

a. Weigh 0.18 g (or 0.36 g) of SeaKem Gold (SKG) agarose into 250 ml screw-cap flask
b. Add 10.0 ml (or 20.0 ml) TE Buffer; swirl gently to disperse agarose.
c. Loosen or remove cap and cover loosely with clear film, and microwave for 30-sec; mix gently and repeat for
10-sec intervals until agarose is completely dissolved.
d. Recap flask and place in 55± 2°C water bath and equilibrate the agarose in the water bath for 15 minutes or
until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: SeaKem Gold agarose works well for making PFGE plugs because it provides added strength to
the plugs minimizing breakage of plugs during the lysis and washing steps. The time and
temperature needed to completely dissolve the agarose is dependent on the specifications of the
microwave used, and will have to be determined empirically in each laboratory.

6. Take out tubes of Lysozyme (20 mg/ml) and Mutanolysin (5U/µl) needed from the -20ºC freezer.

7. Place the 2X Cell Lysis Buffer in a 37± 2°C water bath and the PIV buffer on ice.

8. Remove the TPGY cultures from the anaerobic incubator and pipet the entire 10 ml into labeled Nalgene 50 ml
centrifuge tubes and keep on ice.

9. Centrifuge at 1500 × g for 15 minutes at 4°C.

10. Prepare a master mix containing diluted formaldehyde according to the following table. Prepare enough for
each sample plus one additional aliquot.

Reagent μl/Plug Slice

PIV buffer 3.6 ml

Formaldehyde (35-40%) 400 μl

VERSION: REPLACED BY: AUTHORIZED BY:

Page 14 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

Note: This step should be done using a chemical fume hood.

11. Remove the Nalgene tubes containing the bacterial pellets from the centrifuge and decant the supernatant into a
waste container containing bleach.

12. Gently resuspend each bacterial pellet with 4 ml of PIV + formaldehyde and place on ice for 1 hour with gentle
shaking every 15 minutes.

Note: Steps 11 and 12 should be performed in a Class 2 biological safety cabinet.

13. Centrifuge at 1500 × g for 15 minutes at 4°C.

14. Decant the supernatants into a waste container in a chemical fume hood.

15. Resuspend each bacterial pellet in 4 ml of cold PIV buffer.

16. Repeat step 13 - 15 two more times.

17. Centrifuge at 1500 × g for 15 minutes at 4°C.

18. Decant the supernatants into a waste container in a chemical fume hood.

19. Prepare a master mix containing 2X Cell Lysis Buffer (pre-warmed to 37°C), RNase A, mutanolysin, and
lysozyme according to the following table. Prepare enough for each sample plus one additional aliquot.

Reagent μl/Plug Slice

2X Cell Lysis Buffer 500 μl

RNase (100mg/ml) 0.2 μl

Mutanolsyin 4 μl

Lysozyme 50 μl

20. Resuspend each bacterial pellet in 500 µl of this master mix and transfer to labeled microcentrifuge tubes.

21. Add 500 µl of melted agarose to each cell suspension, mix gently a few times, and immediately transfer to
PFGE plug molds. Agarose suspension is enough to fill two plug molds.

Note: Prewarm the cell suspensions at 55± 2°C for ≥30 seconds prior to adding the agarose.

22. Allow the plugs to solidify in the plug molds at 4± 2°C for at least 15 minutes.

23. Prepare a master mix containing 1X Cell Lysis Buffer (prewarmed to 37°C), RNase A, mutanolysin, and
lysozyme according to the following table. Prepare enough for each sample plus one additional aliquot.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 15 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

Reagent μl/Plug Slice

2X Cell Lysis Buffer 2 ml

Distilled Water 2 ml
RNase (100mg/ml) 0.8 μl

Mutanolsyin 16 μl

Lysozyme 200 μl

24. Pipet 4 ml of this master mix into separate, labeled 50 ml conical tubes for each sample. Fit a green screen cap
onto each 50 ml conical tube and prewarm at 37± 2°C.

25. Remove the plug molds from the refrigerator and using a spatula remove the sample plugs and place into the
corresponding 50 ml conical tubes containing the prewarmed lysis solutions.

26. Place the conical tubes containing the plugs into the shaking water bath at 37± 2°C and incubate overnight
shaking at approximately 60 rpm overnight.

27. Place both sections of the plug mold, spatulas, and scalpel in 10% bleach. Soak them for 1 hour before washing
them.
Day 4

1. Prepare ES buffer by mixing 500 ml of 0.5M EDTA and 50 g of Sarkosyl.

2. Place one bottle of distilled water and two bottles of TE buffer in a water bath at 50± 2°C. One glass bottle
containing distilled water and one glass bottle of TE should be equipped with dispensing apparatuses.

3. Decant the lysis solution from each conical tube containing PFGE plugs into a waste container containing
bleach.

4. Add 4 ml of pre-warmed TE into each 50 ml conical tube containing plugs and decant the TE into a waste
container containing bleach.

5. Calculate the volume of Proteinase K required to yield 0.1 mg/ml final concentration as follows:

(stock conc. of Proteinase K) (X) = (final conc. desired) (final volume)

(stock conc. of Proteinase K) (X) = (0.1 mg/ml) (4 ml)

(0.1 mg/ml) x (4 ml) = volume of Proteinase K needed (ml)/sample

(stock conc. of Proteinase K)

6. Add 4 ml ES buffer and the calculated volume of Proteinase K into each conical tube. Incubate the conical tubes
containing the plugs at 50± 2°C shaking at approximately 60 rpm for 3 hours.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 16 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

7. Repeat steps 3, 4, and 6 one more time.

8. Decant the lysis solution from each conical tube containing PFGE plugs into a waste container containing
bleach.

9. Add 10 ml of pre-warmed distilled water into each 50 ml conical tube containing plugs and incubate at 50± 2°C
shaking at approximately 60 rpm for 10 minutes. Decant used distilled water into a waste container containing
bleach.

10. Repeat step 9 one more time.

11. Add 20 ml of pre-warmed TE buffer into each 50 ml conical tube containing plugs and incubate at 50± 2°C
shaking at approximately 60 rpm for 10 minutes. Decant used TE buffer into a waste container containing
bleach..

12. Repeat step 11 four more times.

13. Store the PFGE plugs in the 50 ml conical tubes containing ≥10 ml TE at 4± 2°C until ready for digestion.
Continue with step 1 in "Restriction Digestion" section or store plugs in TE Buffer at 4ºC until needed. Plugs can be
transferred to smaller tubes containing 1 ml of TE for storage.

Day 5

RESTRICTION DIGESTION OF DNA IN AGAROSE PLUGS

Note: A small slice of the plug or the entire plug (made in disposable plug molds) can be digested with the restriction
enzyme. Restriction digestion of a small slice of the plug is recommended because less enzyme is required and other
slices of the plug can be subjected to restriction analysis with other enzymes. Restriction analysis with a secondary
enzyme is important in situations where the PFGE patterns obtained with the primary enzyme from two or more isolates
are indistinguishable.

1. Label 1.5 ml microcentrifuge tubes with sample numbers.

2. Prepare 1X restriction buffer by diluting the appropriate 10X restriction buffer (provided with each restriction
enzyme by the vendor) 1:10 with distilled water according to the following tables:

SmaI digestion buffer

Reagent μl/Plug Slice

Sterile Distilled Water 180 μl

Restriction Buffer 4 20 μl

Total Volume 200 μl

VERSION: REPLACED BY: AUTHORIZED BY:

Page 17 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

XhoI digestion buffer

Reagent μl/Plug Slice

Sterile Distilled Water 178 μl

Restriction Buffer 4 20 μl
100X BSA 2 μl

Total Volume 200 μl

3. Add 200 μl diluted restriction buffer (1X) to the corresponding 1.5 ml microcentrifuge tubes.

4. Carefully remove plug from TE with spatula and cut a 2.0 to 2.5 mm wide slice from test sample plugs with a single
edge razor blade.

Note: The shape and size of the plug slice that is cut will depend on the size of the comb teeth that are used for casting
the gel. PulseNet recommends that the combs with larger teeth (10 mm wide teeth) be used to cast the gels because
computer analysis of the gel lanes is more accurate and less tedious than analysis of gel lanes cast with combs with the
smaller teeth (5.5-mm). The number of slices that can be cut from the plugs will depend on the skill and experience of
the operator, integrity of the plug, and whether the slices are cut vertically or horizontally (plugs made in disposable
molds).

5. Transfer the plug slices into microcentrifuge tube containing appropriate diluted restriction buffer. Be sure plug slice
is under buffer. Replace rest of plug into the original tube that contains TE buffer.

6. Incubate 1.5 ml microcentrifuge tubes containing plugs with restriction buffer for SmaI digestions at 25± 2°C and
restriction buffer for XhoI digestions at 37± 2°C for 1 hour.

7. Prepare restriction enzyme master mixes with the appropriate restriction enzyme per sample according to the
following tables. Prepare enough for each plug slice plus one additional aliquot.

SmaI (20U/μl stock concentration)

Reagent μl/Plug Slice

Sterile Distilled Water 175 μl

10X Restriction Buffer

20 μl
4
Enzyme (100 U/sample) 5 μl

Total Volume 200 μl

VERSION: REPLACED BY: AUTHORIZED BY:

Page 18 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

XhoI (20U/μl stock concentration)

Reagent μl/Plug Slice

Sterile Distilled Water 173 μl

10X Restriction Buffer

20 μl
4
100X BSA 2 μl
Enzyme (100 U/sample) 5 μl

Total Volume 200 μl

8. After incubation, remove buffer from plug slice using a pipet fitted with 200-250 μl tip all the way to bottom of tube
and aspirate buffer. Be careful not to damage the plug slice with pipet tip and that plug slice is not discarded with
pipet tip.

9. Add 200µl of the appropriate restriction enzyme cocktail to each of the tubes, making sure the plug is
completely submerged in the solution. Close the tubes and mix by gently tapping.

10. Incubate 1.5 ml microcentrifuge tubes containing plugs with restriction buffer SmaI at 25± 2°C and with restriction
buffer XhoI at 37± 2°C overnight.

11. Soak the used green screen caps in 10% bleach for at least one hour and rinse with sterile water prior to storage.

Day 6

1. Label 1.5 ml microcentrifuge tubes for Salmonella ser. Braenderup H9812 standards. One standard plug is needed
per every 3-4 samples.

2. Prepare 1X restriction buffer by diluting the appropriate 10X restriction buffer (provided with each restriction
enzyme by the vendor) 1:10 with distilled water according to the following tables:

XbaI digestion buffer

Reagent μl/Plug Slice

Sterile Ultrapure Water 180 μl

Restriction Buffer H 20 μl

Total Volume 200 μl

3. Add 200 μl diluted restriction buffer (1X) to the corresponding 1.5 ml microcentrifuge tubes.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 19 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

4. Carefully remove standard plugs from cryovials containing TE and cut 2.0 to 2.5 mm wide slices with a single edge
razor blade.

5. Transfer the plug slices to tubes containing the diluted restriction buffer. Be sure each plug slice is under the buffer.
Replace the rest of the plug into the cryovial and store at 4oC ± 2oC.

6. Incubate 1.5 ml microcentrifuge tubes containing plugs with restriction buffer for XbaI digestions at 37oC ± 2oC for
10 minutes.

7. Prepare a restriction enzyme master mix with the appropriate restriction enzyme per sample according to the
following table. Prepare enough for each plug slice plus one additional aliquot.

XbaI (20U/μl stock concentration)

Reagent μl/Plug Slice

Sterile Distilled Water 175 μl

10X Restriction Buffer

20 μl
H
Enzyme (100 U/sample) 5 μl

Total Volume 200 μl

8. After incubation, remove buffer from plug slice using a pipet fitted with 200-250 μl tip all the way to bottom of tube
and aspirate buffer. Be careful not to damage the plug slice with pipet tip and that plug slice is not discarded with
pipet tip.

9. Add 200µl of the XbaI restriction enzyme cocktail to each of the tubes, making sure the plug is completely
submerged in the solution. Close the tubes and mix by gently tapping.

10. Incubate 1.5 ml microcentrifuge tubes containing plugs with XbaI at 37oC ± 2oC for 2 hours.

LOADING PFGE PLUG SLICES AND POURING AGAROSE GEL

11. Make 0.5X Tris-Borate EDTA Buffer (TBE) that is needed for both the gel and electrophoresis running buffer by
adding 125 ml of 10X TBE into 2,375 ml distilled water.

Note: Steps 12-24 are repeated for each gel that will be run.

12. Prepare 1% agarose by measuring 1.5 g of SKG Gold Agarose into a 250 ml bottle. Add 150 ml of 0.5X TBE
buffer to the bottle and microwave on high power until completely melted. Transfer the bottle containing 1%
agarose into the water bath set at 55± 2°C.

13. Place the gel mold on a leveling table and adjust until perfectly leveled. Place the comb holder so the front part (side
with small metal screws) and teeth face the bottom of gel frame and the comb teeth are slightly above the surface of
the gel mold.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 20 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

14. Remove all of the restricted plug slices (digested with SmaI, XhoI, or XbaI) from the water baths. Remove
enzyme/buffer mixture and add 200 μl 0.5X TBE. Incubate at room temperature for 5 min.

15. Remove plug slices from tubes; put comb on bench top and load plug slices on the bottom of the comb teeth.

16. Position comb in gel mold and confirm that the plugs slices are correctly aligned on the bottom of the comb teeth
and that the lower edge of the plug slice is flush against the black platform, and there are no bubbles.

17. Carefully pour the agarose (cooled to 55°C ± 2°C) into the gel mold and allow the gel to solidify for a minimum of
15 minutes at room temperature.

18. Put black gel frame in electrophoresis chamber and add 2.2 L freshly prepared 0.5X TBE. Close cover of unit. (The
amount of buffer needed depends on whether residual buffer was left in tubing or if unit was flushed with water
after the last gel was run.)

19. Turn on cooling module (14ºC), power supply, and pump (set at ≈70 to achieve a flow rate of 1 liter/minute).

20. After the gel has solidified, add 5-10 ml of 0.5X TBE to the comb and gently remove from the gel.

21. Unscrew and remove end gates from gel mold; remove excess agarose from sides and bottom of casting platform
with a tissue. Keep gel on casting platform and carefully place gel inside black gel frame in electrophoresis
chamber.

22. Add 860 µl of Thiourea (10mg/ml) to 0.5X TBE in the electrophoresis chamber. Close cover of chamber.

SAFETY WARNING: Thiourea is a toxic chemical. Weigh thiourea in a chemical fume hood; use gloves, eye
protection, and disposable spatula when handling this chemical. Clean up any spills, and wipe down balance and
surrounding area with a moistened towel. Discard gloves, spatula, weighing paper, etc. as hazardous waste,
according to the guidelines of your institution. Re-cap bottle tightly after use.

ELECTROPHORESIS CONDITIONS

1. Select the following conditions on the CHEF MAPPER:

a. Select the following conditions on the CHEF MAPPER

Auto Algorithm
30 kb - low MW
600 kb - high MW
Initial switch time: 0.5 s
Final switch time: 40 s
Change run time to 18 - 20 h (See note below)
(Default values: Initial switch time = 2.16 s; Final switch time = 54.17 s)

b. Select following conditions on CHEF-DR III

Initial switch time: 2.2 s
Final switch time: 54.2 s
Voltage: 6 V
Included Angle: 120°
Run time: 18-20 h (See note below)

VERSION: REPLACED BY: AUTHORIZED BY:

Page 21 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

Day 7

STAINING AND DOCUMENTATION OF PFGE AGAROSE GEL

1. When electrophoresis run is over, turn off equipment; remove and stain gel with ethidium bromide. Dilute 50 μl of
ethidium bromide stock solution (10 mg/ml) with 500 ml of reagent grade water (this volume is for a staining box
that is approximately 14-cm x 24-cm; a larger container may require a larger amount of staining solution). Stain gel
for 20 minutes in covered container with gentile agitation.

Note: Use extreme caution when handling Ethidium Bromide, as it is carcinogenic and a mutagenic chemical. Stock
solutions of 10 mg/ml Ethidium Bromide (EtBr) in water are available from several commercial companies (Amresco
X328; Bio-Rad, 161-0433; Sigma, E-1510). CDC does not recommend disposing of EtBr down the drain. Aqueous
solutions containing EtBr can be filtered through charcoal or degraded using activated carbon destaining or “tea” bags
from Amresco (E732-25 Destaining Bags) or other companies, which effectively and safely remove EtBr from solutions
and gels. Once the EtBr is removed, the treated aqueous solutions can be discarded down the drain. If you have further
questions about EtBr please refer to the Material Safety Data Sheets (MSDS) provided by the vendor or manufacturer.

2. Destain gel in approximately 500 ml reagent grade water for 60 min; changing water every 20 minutes. Capture
image using a Gel Doc 1000, 2000, EQ, or XR, or equivalent documentation system. If too much background is
observed destain for an additional 30-60 min.

a. Alternative Method for PFGE Gel Staining (GelStar): Dilute 40 ul of GelStar (10,000X stock solution) into 400
ml of 1X TBE. Stain gel for 60 minutes in covered container with gentile agitation. Capture image using a Gel Doc
1000, 2000, EQ, or XR, or equivalent documentation system.

Note: Destaining is not necessary when staining the gel with GelStar. After staining, proceed to capturing the image
with a gel documentation system. Stock solutions of GelStar are available from Lonza, 50535. Use the same precautions
when handling and disposing of GelStar as indicated above for EtBr.

3. Follow directions given with the imaging equipment to save gel image as an *.img or *.1sc file; convert this file to
*.tif file for analysis with the BioNumerics software program. The gel image should fill the entire window of the
imaging equipment (computer) screen (without cutting off wells or lower bands). Ensure that the image is in focus
and that there is little to no staturation (over-exposure) in the bands. Additional instructions are provided in PNL07
of the PulseNet QA/QC manual.

4. Drain buffer from electrophoresis chamber and discard. Rinse chamber with 2-4 L reagent grade water or, if unit is
not going to be used for several days, flush lines with water by letting pump run for 5-10 min before draining water
from chamber and hoses. Remove and clean loose pieces of agarose in the electrophoresis chamber.

5. If the lowest band in the H9812 standard does not migrate within 1 -1.5 cm of the bottom of the gel, the run time
will need to be determined empirically for the conditions in each laboratory.

Use of trade names and commercial sources is for identification purposes only and does not imply endorsement by CDC
or the U.S. Department of Health and Human Services.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 22 of 23
 STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CLOSTRIDIUM CODE: PNL25
Effective Date:
BOTULINUM 1 25 2012

NOTE: CLIA LABORATORY PROCEDURE MANUAL REQUIREMENTS

Efforts have been made to assure that the procedures described in this protocol have been written in accordance with the
1988 Clinical Laboratory Improvement Amendments (CLIA) requirements for a procedure manual (42 CFR 493.1211).
However, due to the format required for training, the procedures will require some modifications and additions to
customize them for your particular laboratory operation.

Any questions regarding the CLIA requirements for a procedure manual, quality control, quality assurance, etc., should
be directed to the agency or accreditation organization responsible for performing your laboratory's CLIA inspection. In
addition, some states and accreditation organizations may have more stringent requirements that will need to be
addressed.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

7.1 Carolina Lúquez, PhD

Botulism Outbreak Investigations Unit
National Botulism Laboratory Team
Centers for Disease Control and Prevention
E-mail: [email protected]
Phone: (404) 639-0896

8. AMENDMENTS:
8.1 1/25/12 the line “or 50 ml of a 10% Sarkosyl Solution” was removed from the Cell Lysis Buffer
Preparation section under Day 3 step 3. This leaves only the 5g Sarkosyl in order to make the final
concentration 5% Sarkosyl and the final volume as 100 ml.
8.2 10/2012 An alternative procedure was added beginning on page 13 of this document.
8.3 5/2/2013 XhoI digestion buffer amounts were corrected under Day 4 step 2 (page 7).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 23 of 23
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

1. PURPOSE: to describe the standardized laboratory protocol for molecular subtyping of Shiga
toxin-producing Escherichia coli O157 (STEC O157) and Salmonella enterica serotypes
Typhimurium and Enteritidis.

2. SCOPE: to provide the PulseNet participants with a single protocol for performing MLVA of
STEC O157 and Salmonella serotypes Typhimurium and Enteritidis, thus ensuring inter-laboratory
comparability of the generated results.

3. DEFINITIONS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 VNTR: Variable-number tandem repeat
3.3 DNA: Deoxyribonucleic acid
3.4 DNase: Deoxyribonuclenase
3.5 PCR: Polymerase chain reaction
3.6 HPLC: High purity liquid chromatography
3.7 dNTP: Deoxyribonucleotide triphosphate
3.8 CDC: Centers for Disease Control and Prevention
3.9 SOP: Standard Operating Procedure

4. RESPONSIBILITIES/PROCEDURE
4.1. Biosafety warning: STEC O157 and Salmonella serotypes Typhimurium and Enteritidis
with an infectious dose as low as 100 cells is a human pathogen capable of causing
serious disease. Always use a minimum of Biosafety level 2 practices and extreme
caution when transferring and handling strains of these serotypes. Work in a biological
safety cabinet when handling large amounts of cells. Disinfect or dispose of all plastic
ware and glassware that come in contact with the cultures in a safe manner.
4.2. Reagents, supplies and equipment needed for DNA template preparation
4.2.1 Trypticase soy agar with 5 % sheep blood (TSA-SB) or comparable media
4.2.2 1 µl inoculation loops
4.2.3 0.5 ml microcentrifuge tubes
4.2.4 DNase-free, molecular biology -grade water
4.2.5 Vortex
4.2.6 Boiling water bath or thermocycler/thermal block accommodating 0.5 ml tubes
4.2.7 Tabletop centrifuge for high rpm (up to 13,000-14,000 rpm) spinning
4.2.8 Pipets (200 µl) for aliquoting 100 µl of DNase-free, molecular biology-grade water
4.2.9 Filtered Sterile Pipet tips
4.3. Reagents, supplies and equipment needed for PCR
4.3.1 DNA templates from isolates (keep at -20oC or -80ºC freezer for long term)
4.3.2 PCR primers (see appendix PNL28-1)
4.3.2.1 Fluorescent-labeled forward primers
4.3.2.1.1 HPLC-purified
4.3.2.2 Unlabeled reverse primers
VERSION: REPLACED BY: AUTHORIZED BY:

1 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.3.2.2.1 Regular gel filtration purification

4.3.2.3 Biosearch Technologies (Novato, CA; www.biosearchtech.com; 1-800-436-
6631) sells primers labeled with the three dyes needed for the protocol
4.3.2.4 Divide the concentrated stocks (100 µM) in portions and store at -80ºC freezer
4.3.2.4.1 One vial should contain enough to prepare 25-50 µl of working solution.
Avoid repetitive freeze-thaw cycles of concentrated primer stocks.
4.3.2.5 The 1.0, 2.5, 5.0, 12.5 and 25.0 µM working solutions can be stored at either
-20oC or -80ºC freezer
4.3.2.6 Prepare new working solutions every month or if a significant drop in the
fluorescence level is observed (for instructions refer to PNQ06_MLVA ABI
certification, appendix PNQ06-5)
4.3.3 96-well polypropylene PCR plates (Fisher, Cat. No. 07-200-613) or Microamp PCR
tubes without caps (Life Technologies, Cat. No. N8010533)
4.3.4 8-well strip caps for the polypropylene plate (Fisher, Cat. No. 07-200-639) or
MicroAmp strip caps for the individual tubes (Life Technologies, Cat. No. N8010535)
4.3.5 DNase-free, molecular biology -grade water
4.3.6 1.5 ml Eppendorf microcentrifuge tubes
4.3.7 PCR Nucleotide Mix (ready-to-use dNTP mix containing all four nucleotides; Roche,
Cat. No. 11 814 362 001)
4.3.8 Platinum Taq Polymerase with 50 mM MgCl2 and 10X buffer (Life Technologies, Cat.
No. 10966-034)
4.3.9 PCR Cooling block (VWR International, Cat. No. 62111-762)
4.3.10 DNA Engine (Biorad), GeneAmp (Life Technologies) or similar thermocycler with a
heated lid option and 96-well block format
4.3.11 Parafilm M, 4” width (VWR, Cat. No. 52858-032)
4.3.12 Complete set (1000 µl, 200 µl, 100 µl, 20 µl, 10 µl and 2 µl) of single channel
pipettors for mastermix set-up (“clean set”)
4.3.13 1-10 µl single channel pipettor for adding DNA templates
4.3.14 Filtered tips for pipettors
4.3.15 Microfuge for low (up to 6,000 rpm) rpm spinning
4.4. Reagents, supplies and equipment needed for Genetic Analyzer 3500
4.4.1 DNase-free, molecular biology -grade water
4.4.2 PCR Cooling block (VWR International, Cat. No. 62111-762)
4.4.3 10 l, 100 l, and 1000 l single channel pipettors
4.4.4 10 l and 200 µl multichannel pipettors
4.4.5 Filtered pipette tips
4.4.6 Sterile solution basins
4.4.7 1.5 ml Eppendorf microcentrifuge tubes
4.4.8 96-well polypropylene (non-PCR) V-bottom plate (for dilutions; Fisher Scientific, Cat.
No. 07-200-698)
4.4.9 MicroAmp Optical 96-well reaction plates (Life Technologies, Cat. No. 4306737)
4.4.10 96-well plate base (Life Technologies, Cat. No. 4317237)
VERSION: REPLACED BY: AUTHORIZED BY:

2 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.4.11 Rubber septa for 96-well reaction plates (Life Technologies, Cat. No. 4315933)
4.4.12 96-well plate retainer (Life Technologies, Cat. No. 4317241)
4.4.13 Hi-Di Formamide (Life Technologies, 25 ml Cat. No. 4311320)
4.4.14 GeneFlo 625 DNA size standard – ROX, 800 µl (Chimerx, Cat. No. 3125-02)
4.4.15 GeneScan Fragment Install Standard DS-33 (Life Technologies, Cat. No. 4376911)
4.4.16 DS-33 Matrix Standard Kit (Dye Set G5) (Life Technologies, Cat. No. 4345833)
4.4.17 Multi-Capillary DS-30 (Dye Set D) Matrix Standard Kit (Life Technologies, Cat. No.
4345827)
4.4.17.1 Needed to establish the system dye color spectra for the instrument. Required
when analyzing fragments labeled with FAM, HEX, NED, and ROX.
4.4.17.2 NOTE: DS-33 Matrix Standard (Dye set G5, required for analyzing fragments
labeled with FAM, VIC, NED, PET, and LIZ) and the GeneScan Fragment
Installation Standard are typically installed as default as part of the instrument
installation process. If any of the three standards is missing from the instrument,
you are not going to be able to start the run.
4.4.17.3 In order to install the DS-30 Matrix, follow the instructions of the kit insert and
the “Getting Started Guide”, chapter “Performing a Spectral Calibration”
4.4.18 3500xL Genetic Analyzer 24-Capillary Array 50 cm (Life Technologies, Cat. No.
4404689) or the 3500 Genetic Analyzer 8-Capillary Array (Life Technologies, Cat.
No. 4404685)
4.4.19 Genetic Analyzer Cathode Buffer Container (4 pack), 1X (Life Technologies, Cat. No.
4408256)
4.4.20 Genetic Analyzer Anode Buffer Container (4 pack), 1X (Life Technologies, Cat. No.
4393927)
4.4.21 3500 Conditioning Reagent (Life Technologies, Cat. No. 4393718)
4.4.22 3500 POP7 Performance Optimized Polymer, (384 samples) (Life Technologies, Cat.
No. 4393708)
4.4.23 Parafilm M, 4” width (VWR, Cat. No. 52858-032)
4.4.24 Centrifuge with a microtiter plate rotor
4.4.25 Heating block or thermal cycler accommodating a 96-well plate for denaturation
4.4.26 Microfuge for low (up to 6,000 rpm) rpm spinning
4.5. DNA template preparation
4.5.1 Day 0:
4.5.1.1 Streak an isolated colony from test cultures to TSA-SB plate (or comparable
media). Incubate cultures at 37ºC for 14-18 hrs.
4.5.2 Day 1:
4.5.2.1 For each isolate to be typed, aliquot 100 l of sterile, molecular biology-grade
water into 0.5 ml microfuge tubes. Use a sterile, disposable 1 µl loop to pick 2-3
colonies (about half of a loopful); rotate the loop in the microfuge tube to release
the bacteria into the water. Cap and vortex for 10-15 seconds to disperse any
clumps.

VERSION: REPLACED BY: AUTHORIZED BY:

3 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.5.2.2 Place the tubes in a 99-100ºC water bath or heat block for 10-15 minutes. Cool
briefly on ice or in fridge and centrifuge for 10 minutes at 10,000 rpm. Place on
ice or in fridge while preparing PCR reactions. These DNA templates can be
stored at -20oC or -80ºC for several years.
4.6. PCR procedure
4.6.1 Day 1:
4.6.1.1 Fill out, save with the run name, and an organism specific (copy and paste the
appropriate sizing tables for the controls) MLVA Fragment Analysis ABI
Worksheet (see appendix PNL28-2) with appropriately labeled samples (a
maximum of 46 isolates/plate; 44 unknowns + two positive controls and a
negative control; two wells are reserved for the internal ladder).
4.6.1.1.1 For each isolate, two wells must be labeled as follows: “BNkeyR1” where
“BNkey” represents the isolate-specific state laboratory identification
number (be sure to use the exact same isolate ID that is used in the PFGE
gels uploaded to the national database) and “R1” represents one of the two
specific multiplex PCR reactions (R1, R2).
4.6.1.2 Fill out, and print a PCR mastermix calculation worksheet (see appendices PNL28-
3a, PNL28-3b and PNL28-3c) by typing the number of isolates to be tested (plus
2-3 extra) in the PCR mastermix calculators labeled R1 and R2. This number is
highlighted in RED and is next to “number of samples to be analyzed”. The
mastermixes for reactions 1 and 2 (R1, R2) for one sample are as follows:

STEC O157:H7 (appendix PNL28-3a)

R1 Volume ( µl) Final conc.

PCR water 5.30
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
VNTR-3F (25 µM) 0.27 0.67 µM
VNTR-3R (25 µM) 0.27 0.67 µM
VNTR-34F (5 µM) 0.24 0.12 µM
VNTR-34R (5 µM) 0.24 0.12 µM
VNTR-9F (5 µM) 0.24 0.12 µM
VNTR-9R (5 µM) 0.24 0.12 µM
VNTR-25F (2.5 µM) 0.20 0.05 µM
VNTR-25R (2.5 µM) 0.20 0.05 µM
Taq (5 U/µl) 0.20 1U
= 9.00

R2 Volume (µl) Final conc.

PCR water 5.92
VERSION: REPLACED BY: AUTHORIZED BY:

4 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

PCR buffer (10x) 1.00 1x

MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
VNTR-17F (5 µM) 0.30 0.15 µM
VNTR-17R (5 µM) 0.30 0.15 µM
VNTR-19F (1 µM) 0.16 0.016 µM
VNTR-19R (1 µM) 0.16 0.016 µM
VNTR-36F (1 µM) 0.11 0.011 µM
VNTR-36R (1 µM) 0.11 0.011 µM
VNTR-37F (2.5 µM) 0.07 0.017µM
VNTR-37R (2.5 µM) 0.07 0.017µM
Taq (5 U/µl) 0.20 1U
= 9.00

Salmonella serotype Typhimurium (appendix PNL28-3b)

R1 Volume ( µl) Final conc.

PCR water 3.81
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.45 2.25 mM
dNTPs (10 mM) 0.20 0.20 mM
ST3-F (5 µM) 0.10 0.05 µM
ST3-R (5 µM) 0.10 0.05 µM
ST5-F (25 µM) 0.60 1.50 µM
ST5-R (25 µM) 0.60 1.50 µM
ST7-F (5 µM) 0.80 0.40 µM
ST7-R (5 µM) 0.80 0.40 µM
ST10-F (2.5 µM) 0.12 0.03 µM
ST10-R (2.5 µM) 0.12 0.03 µM
Taq (5 U/µl) 0.30 1.50 U
= 9.00

R2 Volume (µl) Final conc.

PCR water 4.38
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.32 1.60 mM
dNTPs (10 mM) 0.20 0.20 mM
ST2-F (25 µM) 0.36 0.90 µM
ST2-R (25 µM) 0.36 0.90 µM
ST6-F4 (5 µM) 0.56 0.28 µM
ST6-R2 (5 µM) 0.56 0.28 µM
ST8-F3 (5 µM) 0.48 0.24 µM
VERSION: REPLACED BY: AUTHORIZED BY:

5 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

ST8-R2 (5 µM) 0.48 0.24 µM

Taq (5 U/µl) 0.30 1.50 U
= 9.00

Salmonella serotype Enteritidis (appendix PNL28-3c)

R1 Volume (µl) Final conc.

PCR water 4.91
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
SE1-F (2.5 µM) 0.20 0.05 µM
SE1-R (2.5 µM) 0.20 0.05 µM
SE2-F (12.5 µM) 0.32 0.40 µM
SE2-R (12.5 µM) 0.32 0.40 µM
SE8-F (2.5 µM) 0.28 0.07 µM
SE8-R (2.5 µM) 0.28 0.07 µM
SE6-F (12.5 µM) 0.34 0.43 µM
SE6-R (12.5 µM) 0.34 0.43 µM
Taq (5 U/µl) 0.20 1.00 U
= 9.00

R2 Volume (µl) Final conc.

PCR water 5.52
PCR buffer (10x) 1.00 1x
MgCl2 (50 mM) 0.40 2.00 mM
dNTPs (10 mM) 0.20 0.20 mM
SE5-F (2.5 µM) 0.20 0.05 µM
SE5-R (2.5 µM) 0.20 0.05 µM
SE3-F (12.5 µM) 0.40 0.50 µM
SE3-R (12.5 µM) 0.40 0.50 µM
SE9-F (2.5 µM) 0.08 0.02 µM
SE9-R (2.5 µM) 0.08 0.02 µM
Taq (5 U/µl) 0.20 1.00 U
= 9.00
4.6.1.2.1. NOTE: these primer concentrations serve as a starting point. Since
laboratory-specific factors, such as the age of the primer stocks, calibration
status of the thermocyclers and pipettes, etc., affect amplification
efficiency, each laboratory will have to re-optimize the primer
concentrations for optimal detection of all targets. However, any other
parameters stated in the SOP should not be changed.
VERSION: REPLACED BY: AUTHORIZED BY:

6 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.6.1.3 Thaw all reagents and supplies needed for PCR reactions and place on ice; keep
primers light protected as much as possible
4.6.1.3.1 NOTE: PCR mastermixes should be set up in a clean hood that is dedicated
just for this purpose and where no cultures or DNA are handled.
4.6.1.4 Prepare the two separate PCR mastermixes in 1.5 ml Eppendorf tubes following
the instructions in the PCR mastermix calculation worksheet (see appendices
PNL28-3a, PNL28-3b and PNL28-3c). Keep the mastermix on ice while
preparing. Add the mastermix components in the following order: water, 10x
PCR buffer, Mg2Cl, dNTPs, primers, and then finally Taq polymerase. Mix the
reaction mixture by vortexing briefly.
4.6.1.4.1 NOTE: All components except Taq polymerase should be vortexed
thoroughly before adding to the mastermix. Taq may be briefly centrifuged
with low rpm, if necessary, to pull the enzyme down to the bottom of the
tube.
4.6.1.5 Place a 96-well PCR plate or required number of PCR tubes in a PCR cooling
block.
4.6.1.6 Dispense 9.0 l of each mastermix into the appropriate rows of the 96-well
polypropylene plate / PCR tubes as noted in the MLVA Fragment Analysis ABI
Worksheet (see appendix PNL28-2).
4.6.1.7 Add 1 l of PCR water to each of the two different wells representing the
negative controls of the two reactions.
4.6.1.8 Add 1.0 l of DNA template to each of the two different wells representing the
two PCR reactions for each isolate to be tested.
4.6.1.9 Add the positive controls (it is recommended to run the positive control as a
duplicate).
4.6.1.9.1 Use STEC O157 strain EDL933 (ATCC 43895) as a positive control. The
internal ladder to be used will be comprised of pooled PCR products of the
isolates EC04PN0139 and EC04PN0570 (see appendix PNL28-4 for
instructions for ladder preparation).
4.6.1.9.2 Use S. enterica serotype Typhimurium strain LT2 (ATCC 29946) as a
positive control. The internal ladder to be used will be comprised of pooled
PCR products of the isolates CDC_2009K0825 and CDC_2009K0826 (see
appendix PNL28-4 for instructions for ladder preparation).
4.6.1.9.3 Use S. enterica serotype Enteritidis strain K1891 (ATCC25928) as a positive
control. The internal ladder to be used will be comprised of pooled PCR
products of the isolates H9560 and 2010K0017 (see appendix PNL28-4 for
instructions for ladder preparation).
4.6.1.10 Cover all wells / tubes with 8-well strip caps and firmly clamp down to avoid any
evaporation during PCR amplification.
4.6.1.11 Recommendation: briefly spin down the plate / tubes to remove any air bubbles.
4.6.1.12 Program and save the following two PCR cycling conditions:

VERSION: REPLACED BY: AUTHORIZED BY:

7 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

STEC O157:H7 and Salmonella serotype Enteritidis

“O157-SEMLVA”
* 95ºC for 5 min Step 1
* 94ºC for 20 sec Step 2
* 65ºC for 20 sec Step 3
* 72ºC for 20 sec Step 4
* Go to step 2, 34x Step 5
* 72ºC for 5 min Step 6
* Indefinite hold at 4ºC Step 7

Salmonella serotype Typhimurium

“STMLVA”
* 95ºC for 5 min Step 1
* 94ºC for 20 sec Step 2
* 63ºC for 20 sec Step 3
* 72ºC for 20 sec Step 4
* Go to step 2, 34x Step 5
* 72ºC for 5 min Step 6
* Indefinite hold at 4ºC Step 7

4.6.1.12.1 NOTE: Make sure to use the heated lid option on the PCR block and
tube (calculated) temperature control.
4.6.1.13 When the PCR is complete store the amplification products light-protected at 4ºC
until ready to run on the sequencer. If the fragment analysis is not performed the
same day, the plate should be stored at -20oC or -80ºC. The PCR products are
stable for approximately one month, when stored frozen.
4.7 Initial setup of Genetic Analyzer 3500 instrument before the first run:
NOTE: step 4.7 only needs to be performed before the very first run. In future runs
continue directly to step 4.8.
4.7.1 Click on the “3500” icon to open the 3500 Data Collection Software. The software will
open a window and the main screen will display the “Dashboard”. If the connections
are functioning properly, the status light on the sequencer will be green, the “State” of
the instrument will be “Idle”, and all of the consumables should have the number of
days/samples remaining for each item.
4.7.1.1 NOTE: Prior to the first run, but after capillary array installation, the GeneScan
Fragment Install Standard DS-33, DS-33 and DS-30 matrix standards must be
installed. In order to run the DS-30 dye matrix standard, the “dye set D”
parameters must be set up in the Library under the submenu “Dye Set”. To do
this, click “Create” in the toolbar at the top of the window. In the new window,
name the new dye set “D” and from the “dye” drop-down menu select “Any
VERSION: REPLACED BY: AUTHORIZED BY:

8 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

Dye”. Uncheck the boxes (not shaded in gray) for the Purple and Orange dyes,
and click “Save” at the bottom of the window.
4.7.2 Setting up the Size Standard
4.7.2.1 Click on the “Library” icon at the top right of the screen.
4.7.2.2 At the left-hand side of the screen select the “Size Standards” menu option.
4.7.2.3 At the top of this screen select “Create” in the toolbar.
4.7.2.4 A new window will appear and in the box next to “Size Standard” enter in
“GeneFlo625”.
4.7.2.5 From the “Dye Color” drop-down menu select “Red”.
4.7.2.6 In the “Enter new Size Standard definition” box enter the fragment sizes for each
peak (refer to the GeneFlo 625 product insert for the peak sizes). Separate each
size using a comma.
4.7.2.7 When finished, click the “Add Size(s)” button, then click “Save” and “Close”
4.7.3 Setting up Run Parameters:
4.7.3.1 Once the fragment sizes have been assigned the Assay, File Name Conventions,
and Results Group will need to be set up.
4.7.3.1.1 Assay
4.7.3.1.1.1 At the left-hand side of the screen select the “Assays” menu option and
click “Create” in the toolbar at the top of the screen.
4.7.3.1.1.2 For the “Assay Name” enter “Fragtest” and from the “Application Type”
drop-down menu select “Fragment”. Under the “Protocols” heading
make sure “No” is selected for assigning multiple instrument protocols.
4.7.3.1.1.3 Next to “Instrument Protocol” select the “Create New” button. A new
window will appear.
4.7.3.1.1.3.1 From the “Dye Set” drop-down menu select “D”.
4.7.3.1.1.3.2 For the “Run Module” select “FragmentAnalysis50_POP7xl”.
4.7.3.1.1.3.3 An alert window will pop up asking if you would wish to assign a
protocol name. Click “Yes” and next to the “Protocol Name”
replace the ‘_1’ with ‘_2’ (i.e. - FragmentAnalysis50_POP7xl_2).
Use the default values for all other parameters. This will create a
new protocol with the specified criterion.
4.7.3.1.1.3.3.1 NOTE: These are the instrument default running conditions
for Fragment Analysis with a 50 cm capillary array and
POP7 polymer. You can check the running conditions by
clicking the “Library” button at the top right of the
dashboard and then selecting the “Assays” menu option on
the left-hand side of the screen. The default conditions are:
* Oven_Temperature: 60oC
* PreRun_Voltage: 15.0 kVolts
* Pre-Run-Time: 180 sec.
* Injection_Voltage: 1.6 kVolts
* Injection_Time: 15 sec.
VERSION: REPLACED BY: AUTHORIZED BY:

9 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

* Data_Delay_Time: 1 sec.
* Run_Voltage: 19.5 kVolts
* Run time: 1330 sec.
4.7.3.1.1.3.4 When finished, click “Save to Library” and then click “Yes” in the
pop-up window that asks to apply the changes to the assay. Click
“OK” to confirm, and then close this window.
4.7.3.1.1.4 Next to the “Sizecalling Protocol” option select the “Create New”
button.
4.7.3.1.1.4.1 In the new window enter “MLVAsizecalling” as the “Protocol
Name”.
4.7.3.1.1.4.2 From the “Size Standard” drop-down menu select “GeneFlo625”
4.7.3.1.1.4.3 Input the following analysis settings:
* Analysis: Full Range
* Sizing: Partial Sizing
* Start Size: 50
* Stop Size 625
* Size Calling Method: Local Southern Method
* Primer Peak: Present
* Minimum Peak Height:
* Blue: 600
* Green: 600
* Yellow: 600
* Red: 20
* Uncheck Purple and Orange
* Smoothing: none
* Baseline window: 51 pts
* Min. Peak Half Width: 2 pts
* Peak Window Size: 21 pts
* Polynomial Degree: 2
* Slope Threshold Peak Start: 0.0
* Slope Threshold Peak End: 0.0
4.7.3.1.1.4.4 Click on “Save to Library” and “Yes” to apply to the assay. Select
“OK” to confirm and close this window when finished.
4.7.3.1.1.5 Once all protocols have been established for the new assay, click “Save”
and close the window to return to the “Library” main screen.
4.7.3.1.2 File Name Conventions
4.7.3.1.2.1 Access the “File Name Conventions” menu on the left-hand side of the
screen. In the toolbar at the top of the screen select “Create”.
4.7.3.1.2.2 In the window that appears enter “MLVA” next to the “Name” option.
4.7.3.1.2.3 Use the default settings and click “Save” and close the window.
4.7.3.1.3 Results Groups

VERSION: REPLACED BY: AUTHORIZED BY:

10 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.7.3.1.3.1 Access the “Results Groups” menu on the left-hand side of the screen. In
the toolbar at the top of the screen select “Create”.
4.7.3.1.3.2 In the window that appears, enter “MLVA1” as the name for this results
group.
4.7.3.1.3.2.1 When the results group reaches its upper size limit set up a new
results group with a different name (MLVA2, MLVA3…).
4.7.3.1.3.3 Under the “Delimiters” section heading click the “Add” button to insert a
dash in the “Selected Attributes” box on the right-hand side of the
window.
4.7.3.1.3.4 From the “Available Attributes” list, scroll down to the “Plate Name”
option. Select it and click the “Add” button in between the two boxes to
add it to the “Selected Attributes” list.
4.7.3.1.3.5 Uncheck the “Include an Instrument Run Name folder” and “Include an
Injection folder” options, keeping all other default parameters.
4.7.3.1.3.6 Save the results group to the library and close this window.
4.8 Genetic Analyzer 3500 instrument preparation before each run
4.8.1. Day 1
4.8.1.1 Make sure the service console is open and the status light on the sequencer is
green. Make sure a capillary array is installed in the instrument. For installation,
follow the instructions of the “Install Capillary Array” wizard. You can find the
wizards by clicking on the “Maintenance” option in the toolbar at the top right of
the screen and then clicking on the “Maintenance Wizards” menu option in the
left-hand panel. Spectral and spatial calibrations will need to be run for each array
installation. Before setting up a run, check to make sure that the consumables are
not expired.
4.8.1.1.1 NOTE: The RFID tags on the packaging of the consumables automatically
log in the hours spent on the instrument. The run will not start with expired
reagents.
4.8.1.2 Go back to the Dashboard screen by clicking the “Dashboard” button at the top
left of the screen
4.8.1.3 Select the “Create New Plate” icon at the top of the screen. Name the run
following the standardized PulseNet naming system: use the unique identifier
code that was assigned to your laboratory by PulseNet for the first two to four
letters of the file name. The next two spaces will indicate the year and the next
four spaces will indicate the month and the date the run was performed. For
example GA070426 is a run made at the GA Public Health Laboratory on April
26, 2007. If several runs are performed the same day, separate the file names by
using sequential numbers, for example GA070426-1, GA070426-2.
4.8.1.4 From the “Plate Type” drop-down menu, select “Fragment”.
4.8.1.5 Type in the Owner name or initials.
4.8.1.6 Use the default settings (50cm array, POP-7 polymer) for the remaining options
and click on the “Assign Plate Contents” button at the bottom of the screen.
VERSION: REPLACED BY: AUTHORIZED BY:

11 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.8.1.7 The plate view screen will appear

4.8.1.8 Here you can type in the sample IDs or import a sample plate file from a template
(see Appendix PNL28-5 for instructions on importing plate .txt files).
4.8.1.9 To assign the Assay to the plate, proceed as follows:
4.8.1.9.1 Click on “Add from Library” in the “Assays” box at the bottom of the screen.
4.8.1.9.2 In the new window select “Fragtest” from the list of assays.
4.8.1.9.3 Add this assay by clicking on the “Add to Plate” button, and then close the
window.
4.8.1.10 Adding the File Name Conventions (MLVA) and Results Groups (MLVA1) can
be done in the same manner, by selecting the “Add from Library” link in the
respective boxes.
4.8.1.11 Once the assay, file name convention and results group have been added to the
plate, select the entire sample ID set by highlighting the individual cells or by
clicking on column/row headings.
4.8.1.12 Check the box next to each of the run parameters: Assay, File Name Convention,
and Results Group.
4.8.1.13 When each of the run parameters have been successfully assigned a dot will
appear in the center of the cells in which the parameters were applied.
4.8.1.13.1 NOTE1: If a plate layout has been imported from a .txt file the Assay, File
Name Convention and Results Group should automatically be applied to
the plate and check-marked for all samples.
4.8.1.13.2 NOTE2: What identifiers are shown in the sample ID cells can be changed
by selecting or deselecting options in the “Show In Wells” drop-down
menu above the sample plate layout. Assays, file name conventions, and
results groups can be color coded by double-clicking the black dot to the
left of the file name or during initial setup.
4.8.1.14 Save the plate by clicking the “Save Plate” button in the toolbar at the top of the
screen and selecting “Save” from the drop-down menu.
4.8.1.15 Go back to the dashboard screen by clicking the “Dashboard” button in the top
left corner of the screen and continue with fragment analysis preparation.
4.8.1.16 If needed, install the POP7 polymer in the instrument.
4.8.1.16.1 Click on the “Maintenance” button in the toolbar at the top right of the
window, and then select the “Maintenance Wizards” menu option on the
left-hand side.
4.8.1.16.2 Follow the instructions of the “Replenish Polymer” wizard.
4.8.1.17 If needed, also install new anode and cathode buffer containers.
4.8.1.17.1 NOTE1: If an old polymer pouch (been on the instrument > 7 days) is
switched to a new one, follow the instructions of the “Wash Pump and
Channels” wizard instead. This process takes 30 minutes to complete and
requires a conditioning pouch.

VERSION: REPLACED BY: AUTHORIZED BY:

12 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.8.1.17.2 NOTE2: To update the system after new reagents have been installed, click
the “Refresh” button under the “Consumables Information” heading on the
Dashboard.
4.9. Fragment analysis sample preparation
4.9.1 Day 1
4.9.1.1 NOTE: The fragment analysis method is not organism specific therefore; STEC
O157 and Salmonella serotypes Typhiumurium and Entertidis may be run on a
single fragment analysis plate.
4.9.1.2 Thaw the Hi-Di Formamide, the GeneFlo 625 DNA size standard and the internal
ladder (see appendix PNL28-4) and place on ice.
4.9.1.2.1 NOTE: aliquot Hi-Di Formamide (500 µl / tube) and the size standard (50
µl / tube) in order to avoid frequent freeze-thaw cycles.
4.9.1.3 Prepare a 96-well V-bottom plate for diluting the PCR reactions. Using a 200 µl
multichannel pipettor and a solution basin, dispense 19 µl of molecular-grade
water in the required number of wells.
4.9.1.4 Remove the plate / tubes with the PCR reactions from the thermocycler. Briefly
spin down the plate / tubes, if necessary. Use a 10 µl multichannel pipettor to
transfer 1 µl of each PCR reaction directly across to the corresponding set of
wells in the dilution plate. In order to avoid cross-contamination, remove the strip
cap from just one column at a time and recap the column before opening the next
one.
4.9.1.5 For the internal ladder, combine R1 and R2 PCR products from the four PCR
reactions of both internal ladder isolates to end up with a total of 40 µl. Mix well
by pipetting up and down a few times and add 3 l of internal ladder in two
wells.
4.9.1.6 Using a 200 µl multichannel pipettor, mix the dilutions by pipetting up and down
a few times. Cover the plate with parafilm and put in the fridge or on ice.
4.9.1.7 Prepare a fragment analysis master mix containing DNA size standard and Hi-Di
Formamide for the samples following the calculations indicated in the table
below. The fragment analysis mastermix calculations can also be performed
using the autocalculate box at the bottom of the MLVA Fragment Analysis ABI
Worksheet (see appendix PNL28-2). Vortex briefly and place on ice.

Reagents Frag. anal. mastermix

Hi-Di Formamide 8 µl x (# samples +3)=
GeneFlo 625 bp size standard 1 µl x (# samples +3)=

4.9.1.8 Place a MicroAmp Optical 96-well sample plate in a cold block. Aliquot 9 µl of
the prepared fragment analysis mastermix to the required number of wells. Cover
the plate loosely with Parafilm.

VERSION: REPLACED BY: AUTHORIZED BY:

13 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.9.1.9 Using the 10 µl multichannel pipettor, add 1 µl of 1:20 diluted PCR reactions to
the appropriate columns in the sample plate. Keep sliding the Parafilm sheet from
column to column to keep track of the sample order.
4.9.1.10 Denaturate templates by heating the reaction plate uncovered in a thermocycler at
95oC for 3 min.
4.9.1.11 While the templates denaturate, turn the oven of the 3500 on using the “Start Pre-
Heat” button located on the middle, right-hand side of the dashboard. The default
set point should be 60°C.
4.9.1.12 Briefly spin down the sample plate to remove any air bubbles.
4.9.1.13 Seal the plate with the rubber septa. Place the sample plate in a plate base. Snap
the plate retainer onto the plate and the plate base.
4.9.1.14 Place the plate assembly and the buffer into the Genetic Analyzer 3500.
4.9.1.14.1 Push the tray button at the front of the Genetic Analyzer 3500 to bring the
autosampler to the forward position. Open the instrument door.
4.9.1.14.2 Place the plate assembly on the autosampler in position A or B with the
notched corner of the plate base facing you.
4.9.1.14.3 Close the instrument door and wait for the green light to illuminate.
4.9.1.14.4 Open the previously created plate setup by clicking the blue “Edit Existing
Plate” button at the top of the dashboard screen.
4.9.1.14.5 Select the plate to be run and click “Open”.
4.9.1.14.6 Link the plate by clicking “Link Plate for Run” at the bottom of the screen
4.9.1.14.7 A “Load Plate” pop-up window will verify that the plate has been
successfully loaded. Click “OK”.
4.9.1.15 The “Start Run” button at the bottom of the screen will become functional
indicating that the run can be started. Make sure the plate is linked to the correct
position (A or B), then click the “Start Run” button. Click “OK” on the alert
window. The run will start.
4.10. Viewing and exporting data from the Genetic Analyzer 3500
4.10.1 Day 2
4.10.1.1 NOTE: Steps 4.10.1.3 and 4.10.1.4 only need to be performed before the very
first analysis.
4.10.1.2 Double-click on the shortcut icon for GeneMapper v4.1 or higher and enter the
appropriate password to access the software. The main menu window will open.
4.10.1.3 Set up the size standard:
4.10.1.3.1 From the “Tools” drop-down menu, select “GeneMapper Manager”. A
“GeneMapper Manager” window will open.
4.10.1.3.2 Select the “Size Standards” tab.
4.10.1.3.3 Click on “New”, leave the default option “Basic or Advanced” selected and
click “OK”. The “Size Standard Editor” window will appear.
4.10.1.3.4 Name the new size standard “GeneFlo625”.
4.10.1.3.5 Leave the default option “Red” as “Size Standard Dye”.

VERSION: REPLACED BY: AUTHORIZED BY:

14 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.10.1.3.6 Enter sizes for each peak in the table (refer to the GeneFlo 625 product
insert for the peak sizes).
4.10.1.3.7 When finished, click “OK”.
4.10.1.4 Set up analysis method:
4.10.1.4.1 From the “Tools” drop-down menu, select “GeneMapper Manager”. A
“GeneMapper Manager” window will open.
4.10.1.4.2 Select the “Analysis Methods” tab.
4.10.1.4.3 Click on “New”, leave the default option “Microsatellite” selected and click
“OK”. The “Analysis Methods Editor” window will appear.
4.10.1.4.4 Name the new method “PNMLVA”.and click “OK”.
4.10.1.4.5 Highlight the new method name “PNMLVA” and click “Open”.
4.10.1.4.6 Select the “Peak Detector” tab and change “Peak Detection Algorithm” to
“Advanced” from the drop-down menu.
4.10.1.4.7 Input the following analysis settings and click “OK” when finished:
*Analysis: Full Range
* Sizing: Partial Sizing
*Start Size: 50
* Stop Size 625
* Smoothing: none
* Baseline window: 51 pts
* Size Calling Method: Local Southern Method
* Peak Amplitude Thresholds:
* B: 600
* G: 600
* Y: 600
* R: 20
* P: 50
* O: 50
* Min. Peak Half Width: 2 pts
* Polynomial Degree: 2
* Peak Window Size: 21 pts
* Slope Threshold:
* Peak Start: 0.0
* Peak End: 0.0
* Leave the “Enable Normalization” option unchecked
4.10.1.4.8 Click “Done” to close the GeneMapper Manager window and return to the
main screen.
4.10.1.5 From the “File” drop-down menu select “Add Samples to Project”.
4.10.1.6 Find the folder containing the data file to be analyzed: My Computer → AB SW
& DATA (D:) → Applied Biosystems → 3500 → Data → MLVA1-
<PlateName>.

VERSION: REPLACED BY: AUTHORIZED BY:

15 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.10.1.7 Highlight the desired file(s) and click on “Add to List”. File(s) will appear in the
window on the right. Click “Add” below the file list to return to the original
screen.
4.10.1.8 Samples in the selected file(s) will be listed in a new window and the “Analyze”
(play) button appears in green color in the toolbar indicating that the files are
ready to be analyzed.
4.10.1.9 Select the size standard GeneFlo625 for the first sample, highlight the size
standard column by selecting the heading, and select “Fill Down” from the “Edit”
drop-down menu.
4.10.1.10 Select the analysis method PNMLVA for the first sample, highlight the analysis
method column, and select “Fill Down” from the “Edit” drop-down menu.
4.10.1.11 Click on the “Play” icon.
4.10.1.12 Name the project with the run name (for example, “GA070426”) and click “OK”.
4.10.1.13 A successful analysis is indicated by green squares. Yellow triangles indicate
problematic components (i.e. missing size standard peaks). Red circles indicate
results fell below acceptable quality values. Samples with yellow or red circles in
the SQ column should be selected for re-analysis.
4.10.1.13.1 To resolve failed analyses due to sub-optimal molecular marker peak
profile (i.e. miscalling of peaks), select a row(s) with a yellow triangle or a
red circle in the SQ column and click on the “Size Match Editor” icon on
the toolbar. The “Size Match Editor” view will appear.
4.10.1.13.2 Place the cursor near the X-axis to activate the magnifying lens, and
then pull up (mouse left-click and hold) to zoom in a specific area to
facilitate editing
4.10.1.13.3 Left-click at the base of a peak to select. Right-click and select
“Add”, “Delete”, or “Change”.
4.10.1.13.4 Select the correct molecular weight for the selected peak from the
drop-down menu. Repeat this process for all other miscalled peaks. Click
“OK” when finished.
4.10.1.13.5 After the size standard has been adjusted, click the “Play” button to
re-analyze the data. After a successful analysis, the samples will have green
squares under the SQ column. If the size standard cannot be adjusted, the
reaction is considered a fragment analysis failure and must be re-run
4.10.1.14 Check the fragment result data (the fluorescent peaks) for each well by
highlighting the well ID and by clicking on the “Display Plots” icon on the
toolbar.
4.10.1.14.1 Make sure that all VNTRs amplified in the positive control and that
the fragment sizes are within the range specified in the appendix PNL28-2
and record the fragment sizes on the MLVA Fragment Analysis ABI
Worksheet.
4.10.1.14.2 The size calling for the internal ladder should also be within the range
specified in the appendix PNL28-2 (or PNL28-4).
VERSION: REPLACED BY: AUTHORIZED BY:

16 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

4.10.1.14.3 Write down any failed reactions in the MLVA Fragment Analysis
ABI Worksheet. Make a note of non-specific bands and primer-dimers.
4.10.1.15 Export the peak file:
4.10.1.15.1 NOTE1: the following column headings should appear in the
exported table in the following order from left to right: “Dye/Sample
Peak”, “Sample File Name”, “Marker”, “Allele”, “Size”, “Height”, “Area
in Point”, “Data Point”. You can modify the format of the table by selecting
the “Table Setting Editor” from the “Tools” drop-down menu. Select the
“Genotypes” tab and make sure that only the boxes for the following are
checked: “Sample File Name”, “Marker”, “Dye”, “Allele”, “Size”,
“Height”, “Area in Point”, and “Data Point”.
4.10.1.15.2 NOTE 2: You can import the peak file also from the Data Collection
Foundation software, but the file format is not correct; the “Allele” and
“Marker” columns are missing and there are additional columns in the file.
This file cannot be imported into BioNumerics without first manually
modifying (adding and deleting appropriate columns) the file.
4.10.1.15.3 Highlight the samples for which you want to export peak data.
4.10.1.15.4 Click on the “Display Plots” icon on the toolbar.
4.10.1.15.5 Click on the “Sizing Table” icon on the toolbar and a table will
appear below the electropherograms.
4.10.1.15.6 From the “File” drop-down menu, select “Export Table”.
4.10.1.15.7 Select the location (for example a flash drive) where you want to
export the data.
4.10.1.15.8 Name the export file with the run name (for example GA070426) and
make sure the file type is tab-delimited text (.txt) file.
4.10.1.16 The remaining gel can stay in the instrument if it is going to be used within 7
days.
4.10.1.16.1 NOTE: To extend life of polymer, remove after run, place in
refrigerator, and replace with an old polymer (on instrument longer than 7
days) until next use. Polymer should not be on instrument for more than a
total of 7 days.

5. FLOW CHART:

6. REFERENCES:
6.1 Hyytiä-Trees, E., Smole, S. C., Fields, P. I.., Swaminathan, B., and Ribot, E. M. (2006)
Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-number
tandem repeat analysis (MLVA) of Shiga toxin-producing Escherichia coli O157 (STEC O157).
Foodborne Pathog. Dis. 3, 118-131.
6.2 Hyytia-Trees, E., Lafon, P., Vauterin, P., and Ribot, E. (2010) Multi-laboratory validation
study of standardized multiple-locus VNTR analysis (MLVA) protocol for Shiga toxin-producing

VERSION: REPLACED BY: AUTHORIZED BY:

17 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC
O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND Effective Date:
ENTERITIDIS– APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 02 26 14
PLATFORM

Escherichia coli O157 (STEC O157): a novel approach to normalize fragment size data between
capillary electrophoresis platforms. Foodborne Path. Dis. 7, 129-136.

7. CONTACTS:
7.1 Eija Trees, D.V.M., Ph.D.
PulseNet Next Generation Subtyping Methods Unit, CDC
(404) 639-3672
[email protected]
7.2 Patti Lafon
PulseNet Next Generation Subtyping Methods Unit, CDC
(404) 639-2828
[email protected]
7.3 Ashley Sabol
PulseNet Next Generation Subtyping Methods Unit, CDC
(404) 639-2947
[email protected]

8. AMENDMENTS:
5/6/2013: former appendix PNL28-4 (BioNumerics specifications for the E. coli O157 VNTR loci)
was moved to SOP PND16 (PulseNet standard operating procedure for analysis of MLVA data of
Shiga toxin-producing Escherichia coli in BioNumerics – Applied Biosystems Genetic Analyzer
3130/3500 data). Former appendices PNL28-5 and PNL28-6 were renamed PNL28-4 and PNL28-5,
respectively.
2/26/2014: the three laboratory SOPs for STECO157 (PNL28), and Salmonella serotypes
Typhimurium (PNL29) and Enteritidis (PNL30) using the ABI 3500 platform were combined into a
single SOP (PNL28).

VERSION: REPLACED BY: AUTHORIZED BY:

18 of 28
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

Appendix PNL28-1

MLVA PCR Primer sequences for STEC O157:H7 and Salmonella serotypes Typhimurium and Enteritidis
Locus Dye1 Forward Primer (5’ to 3’) Reverse Primer (5’ to 3’)
_____________________________________________________________________________________________________________________________ ___________________
STEC O157:H7
VNTR-3 CalRed590 GG CGG TAA GGA CAA CGG GGT GTT TGA ATT G GAA CAA CCT AAA ACC CGC CTC GCC ATC G
VNTR-34 FAM GA CAA GGT TCT GGC GTG TTA CCA ACG G GTT ACA ACT CAC CTG CGA ATT TTT TAA GTC CC
VNTR-9 FAM GC GCT GGT TTA GCC ATC GCC TTC TTC C GTG TCA GGT GAG CTA CAG CCC GCT TAC GCT C
VNTR-25 HEX GC CGG AGG AGG GTG ATG AGC GGT TAT ATT TAG TG GCG CTG AAA AGA CAT TCT CTG TTT GGT TTA CAC GAC
VNTR-17 CalRed590 GC AGT TGC TCG GTT TTA ACA TTG CAG TGA TGA GGA AAT GGT TTA CAT GAG TTT GAC GAT GGC GAT C
VNTR-19 FAM GC AGT GAT CAT TAT TAG CAC CGC TTT CTG GAT GTT C GGG GCA GGG AAT AAG GCC ACC TGT TAA GC
VNTR-36 FAM GG CGT CCT TCA TCG GCC TGT CCG TTA AAC GCC GCT GAA AGC CCA CAC CAT GC
VNTR-37 HEX GC CGC CCC TTA CAT TAC GCG GAC ATT C GCA GGA GAA CAA CAA AAC AGA CAG TAA TCA GAG CAG
C

Salmonella Typhimurium
ST3 HEX GT TCT TCT GCA ACG CAG GCA GAT GGC ATG ACG CTG CAA CG
ST5 FAM TT TTC GCT CAA CAA ACT T ACA GCA CCA GAA GCA AT
ST7 CalRed590 CG ATT GAC GAT ATC TAT GAC TT GTT TTT CAC GTT TGC CTT TC
ST10 HEX CG GGC GCG GCT GGA GTA TTT G GAA GGG GCC GGG CAG AGA CAG C
ST2 FAM CA ACG CCT GTT CAG CAA C ATC AAC AGC GGG TGG AT
ST6 CalRed590 AG CAG TGG CTG GCG GGA AAC C GCA GCC GGA CAG GGG ATA AGC C
ST8 HEX GC AGG TGT GGC TAT TGG CGT TGA AA GAT GGT GAC GCC GTT GCT GAA GG

Salmonella Enteritidis
SE-1 FAM TGT GGG ACT GCT TCA ACC TTT GGG C CCA GCC ATC CAT ACC AAG ACC AAC ACT CTA TGA
SE-2 CallRed590 GTG CTT CCT CAG GTT GCT TTT AGC CTT GTT CG GGG GAA TGG ACG GAG GCG ATA GAC G
SE-8 HEX GGT AGC TTG CCG CAT AGC AGC AGA AGT GGC GGC AAG CGA GCG AAT CC
SE-6 FAM CTG GTC GCA GGT GTG GC GGT GAC GCC GTT GCT GAA GGT AAT AAC AGA GTC
SE-5 HEX GGC TGG CGG GAA ACC ACC ATC GCC GAA CAG CAG GAT CTG TCC ATT AGT CAC TG
SE-3 CallRed590 CGG GAT AAG TGC CAC ATA ACA CAG TCG CTA AGC CGC CAG TGT TAA AGG AAT GAA TGA ACC TGC TGA TG
SE-9 FAM CCA CCT CTT TAC GGA TAC TGT CCA CCA GC GGC GTT ACT GGC GGC GTT CG
_____________________________________________________________________________________________________________________________ ___________________
1
Only the 5’ of the forward primer is fluorescently labeled

VERSION: REPLACED BY: AUTHORIZED BY:

19 of 28
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

20 of 28
LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

VERSION: REPLACED BY: AUTHORIZED BY:

21 of 28
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

22 of 28
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and
autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

23 of 28
 LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

Note: this appendix has been posted on the SharePoint site as an Excel file so that it can be saved locally for data entry and autocalculation

VERSION: REPLACED BY: AUTHORIZED BY:

24 of 28
 5LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

Appendix PNL28-4

Instructions to prepare the internal ladders

1. Prepare DNA templates from isolates as described in the protocol step 4.5. Store the templates at -20oC or -
80oC freezer.
1.1 For STEC O157:H7, use the strains EC04PN0139 and EC04PN0570
1.2 For Salmonella serotype Typhimurium, use strains 2009K0825 and 2009K0826
1.3 For Salmonella serotype Enteritidis, use strains H9560 and 2010K0017
2. Use the DNA templates to set up and run the PCR reactions R1 and R2 as described in the protocol step 4.6.
3. After PCR amplification, pool the R1 and R2 reactions for the two strains into one single PCR tube to end
up with a final volume of 40 µl. Mix by pipetting up and down a few times.
4. A new lot of internal ladder must be tested against the old ladder lot by running them in the same fragment
analysis run.
5. Store the ladder in -20oC or -80oC freezer. It should remain stable at least 5-6 freeze-thaw cycles for a
period of one month.

Expected fragment sizes (bp) of the fifteen fragments (locus VNTR_36 is a null allele in EC04PN0139)
present in the STEC O157:H7 internal ladder as listed in the peak file:
VNTR-36 (B): 170 - 172 NA
VNTR-34 (B): 222 - 224 260 - 262
VNTR-19 (B): 296 - 298 320 - 322
VNTR-9 (B): 518 - 522 565 - 569
VNTR-25 (G): 127 - 128 138 - 140
VNTR-37 (G): 192 - 194 198 - 200
VNTR-17 (Y): 153 - 155 177 - 179
VNTR-3 (Y): 397 - 401 433 - 435

Expected fragment sizes (bp) of the fifteen fragments (locus VNTR_36 is a null allele in EC04PN0139)
present in the STEC O157:H7 internal ladder as they appear in the electropherogram:
VNTR-25 (G): 127 - 128 138 - 140
VNTR-17 (Y): 153 - 155 177 - 179
VNTR-36 (B): 170 - 172 NA
VNTR-37 (G): 192 - 194 198 - 200
VNTR-34 (B): 222 - 224 260 - 262
VNTR-19 (B): 296 - 298 320 - 322
VNTR-3 (Y): 397 - 401 433 - 435
VNTR-9 (B): 518 - 522 565 - 569

VERSION: REPLACED BY: AUTHORIZED BY:

25 of 28
5LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

Expected fragment sizes (bp) of the fourteen fragments present in the Salmonella serotype Typhimurium
internal ladder as listed in the peak file:
ST5 (B): 184 - 185 231 - 233
ST2 (B): 370 - 373 399 - 401
ST3 (G): 177 - 180 188 - 192
ST10 (G): 383 - 384 413 - 415
ST8 (G): 582 - 584 589 - 591
ST7 (Y): 137 - 139 147 - 151
ST6 (Y): 253 - 255 283 - 285

Expected fragment sizes (bp) of the fourteen fragment present in the Salmonella serotype Typhimurium
internal ladder as they appear in the electropherogram:
ST7 (Y): 137 - 139 147 - 151
ST3 (G): 177 - 180 188 - 192
ST5 (B): 184 - 185 231 - 233
ST6 (Y): 253 - 255 283 - 285
ST2 (B): 370 - 373 399 - 401
ST10 (G): 383 - 384 413 - 415
ST8 (G): 582 - 584 589 - 591

Expected fragment sizes (bp) of the thirteen fragments (SE9 has the same allele in both ladder strains)
present in the Salmonella serotype Enteritidis internal ladder as listed in the peak file:
SE9 (B) 181 - 184 181 - 184
SE6 (B): 446 - 447 479 - 482
SE1 (B) 190 - 193 211 - 213
SE5 (G): 201 - 203 218 - 221
SE8 (G): 346 - 350 433 - 436
SE3 (Y): 199 – 203 211 - 215
SE2 (Y): 317.5 - 324 363 - 364

Expected fragment sizes (bp) of the fifteen fragments (SE9 has the same allele in both ladder strains)
present in the Salmonella serotype Enteritidis internal ladder as they appear in the electropherogram:
SE9 (B) 181 - 184 181 - 184
SE1 (B) 190 - 193 211 - 213
SE3 (Y): 199 - 203 211 - 215
SE5 (G): 201 - 203 218 - 221
SE2 (Y): 317.5 - 324 363 - 364
SE8 (G): 346 - 350 433 - 436
SE6 (B): 446 - 447 479 - 482

NOTE: fragment size ranges for the internal ladders are based on multiple independent runs at CDC and
PulseNet Participating Laboratories

VERSION: REPLACED BY: AUTHORIZED BY:

26 of 28
 5LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

Appendix PNL28-5

Steps for Exporting and Importing Plate Set ups on the ABI 3500

Note: the procedure described below will require the sequencer computer to have Microsoft Office. If the
sequencer computer does not have Microsoft Office, the exported file needs to be saved on a flash drive so that
steps 10 through 16 can be performed on a computer with Microsoft Office.

1. On the Dashboard screen click on the “Edit Existing Plate” button.

2. Select “Fragment” from the “Filter” drop-down menu.
3. Find the most recent full plate. Click on the ID to highlight the desired plate and then click the “Open”
button at the bottom of the window.
3.1. NOTE: If you do not have a full plate, create a new one by filling in each sample ID space (A01
through H12) with “test” and saving it with the plate name “MLVA_Template”. Be sure to add the
results group “MLVA1”, file name convention “MLVA”, and Assay “Fragtest” to each sample, making
sure each one is check-marked. It is necessary to have all required wells filled in when exporting so that
they will be available in your exported file.
4. In the plate layout window, click the “Export…” button in the toolbar at the top of the screen.
5. In the export window, create a new folder on the desktop by clicking the “Desktop” icon on the left side of
the window. Find the ‘New Folder’ button in the toolbar at the top of the window and name the new folder
“MLVA Plate Setup”.
6. Save the file to this folder by using your new plate name (e.g. - CDC110726) and clicking “Save”.
7. A window will pop up letting you know that the plate has been successfully exported. Click “OK”.
8. Click the “Close Plate” button in the toolbar at the top of the plate layout screen, and click “Yes” to close
without saving changes.
9. Minimize the 3500 program window and open the “MLVA Plate Setup” folder on the desktop.
10. Right-click your plate name, select “Open With” and in the submenu select Microsoft Office Excel.
11. Sample IDs can be typed or copy and pasted from a separate Excel file into the spaces next to the correct
wells under the “Sample Name” heading.
11.1. NOTE: Do not change/delete any of the column headings. The fields must be in the same format
when importing as they were when exported. Additionally, the software will not import the file if the
sample IDs contain special characters (e.g. - ! , / , ) , etc). You can use underscores and dashes.
12. The “Plate Name” should be changed to match the plate/file name, and initials should be placed under the
‘Owner Name’ heading. The defaults for “Application Type”, “Capillary Length (cm)”, “Polymer”, and
“Number of Wells” should be used.
13. Once all of the IDs are inserted, make sure that for each sample ID and all controls, the assay is “Fragtest”,
the results group name is “MLVA1”, the file name convention is “MLVA”, and “Sample” is listed under
“Sample Type”.
14. Under the “File” drop-down menu select “Save”.
15. A warning window will appear asking if the workbook should be saved in the “Text (tab-delimited)” format.
Click “Yes”.
16. Exit Microsoft Excel, clicking “No” when prompted to save changes.
16.1. NOTE: The removal of additional spaces from lines in the .txt file after modification in Excel may be
necessary before importing into the “3500” program. To do this, open the file in “Notepad” making
VERSION: REPLACED BY: AUTHORIZED BY:

27 of 28
 5LABORATORY STANDARD OPERATING PROCEDURE FOR PULSENET CODE: PNL28
MLVA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC Effective Date:
O157) – APPLIED BIOSYSTEMS GENETIC ANALYZER 3500 PLATFORM 02 26 14

sure the window is opened in full screen. In lines 1, 2, and 4 remove any additional spaces after the last
character in the line or from the left side of the page. Once all spaces have been removed from those
lines, save the file by clicking “Save” in the “File” drop-down menu and close the window.
17. Go to the ABI 3500 plate layout window by clicking “Create New Plate” on the Dashboard.
18. Enter in the name of the plate and from the “Plate Type” drop-down menu select “Fragment”.
19. Click on the “Assign Plate Contents” button at the bottom of the screen.
20. In the toolbar at the top of the “Plate View” screen, click the “Import...” button.
21. In the import window, navigate to the recently created plate under Desktop/MLVA Plate Setup. Select the
appropriate .txt file and click “Open”.
21.1. At this point an error message may appear asking if you would like to proceed. If you encounter this
message, click the “Proceed” button at the bottom of the window.
22. When the file has been imported, an alert window will appear stating that the plate “has been successfully
imported”. Click “OK”.
23. In the toolbar at the top of the screen click on “Save Plate” and from the drop-down menu select “Save As”.
Enter the plate name matching the name of the .txt file, and click “OK”.
23.1. NOTE: When importing a plate layout from a .txt file the Assay, File Name Convention and Results
Group should automatically be applied to the plate and check-marked for all samples. If the .txt file
does not contain the correct assay file name (e.g. – the name is misspelled, etc.), the assay will not be
imported and the file name convention and results group will be imported but not check-marked.
23.2. NOTE2: For repeated use, plate templates can be created by exporting a plate to the “MLVA Plate
Setup” folder. Save the file as a tab-delimited text file with “test” as the sample ID and with a new plate
name (e.g. - “MLVA_Template”). After you have filled out the information for a new run on the
template, save it in step 14 with the standardized run name (name (e.g. - CDC101020).

VERSION: REPLACED BY: AUTHORIZED BY:

28 of 28
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

1. PURPOSE: To describe the One-Day (24-26 h) Standardized Laboratory Protocol for Molecular
Subtyping of Cronobacter species by Pulsed-field Gel Electrophoresis (PFGE).

2. SCOPE: To provide the PulseNet participants with a standardized procedure for performing PFGE
of Cronobacter spp., thus ensuring inter-laboratory comparability of the generated results.

3. DEFINITIONS/TERMS:
3.1 PFGE: Pulsed-field Gel Electrophoresis
3.2 DNA: Deoxyribonucleic acid
3.3 CDC: Centers for Disease Control and Prevention
3.4 CLRW: Clinical Laboratory Reagent Water

4. RESPONSIBILITIES/PROCEDURE:

BIOSAFETY WARNING: Cronobacter species are human pathogens and can cause serious disease. Always use
Biosafety Level 2 practices (at a minimum) and extreme caution when transferring and handling strains of this genus.
Disinfect or dispose of all plasticware and glassware that come in contact with the cultures in a safe manner.

Please read all instructions carefully before starting protocol. Treat all plasticware, glassware, pipets, spatulas, etc. that
come in contact with the cell suspensions or plugs as contaminated materials and dispose of, or disinfect according to the
guidelines of your institution. Disinfect reusable plug molds before they are washed; the disposable plug molds,
including the tape and the tab that is used to push the plugs out of the wells, are also contaminated and should be
disinfected 1% Lysol/Amphyll or 90% ethanol for at least 30 minutes if they will be washed and reused.

Day 0
Streak an isolated colony from test cultures onto Trypticase Soy Agar with 5% defibrinated sheep blood (TSA-SB)
plates (or comparable non-selective media) for confluent growth. It is recommended that a storage vial of each culture be
created. To do this stab small screw cap tubes of TSA, HIA, or similar medium with the same inoculating loop used to
streak the plate. This will ensure that the same colony can be retested if necessary. Incubate cultures at 37ºC for 14-18 h.

Day 1
1. Turn on shaker water bath or incubator (54-55ºC), stationary water baths (55-60ºC) and spectrophotometer
(or equivalent instrument such as the Dade Microscan Turbidity meter or bioMérieux Vitek colorimeter).

2. Prepare TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0)1 as follows:

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure Clinical Laboratory Reagent Water (CLRW)

Note: The TE Buffer is used to make the plug agarose and also to wash lysed PFGE plugs.

1
Additional information is found on pages 11 and 12 of this document.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 1 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

3. Prepare 1% SeaKem Gold agarose in TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) for PFGE plugs as follows:
a. Weigh 0.50 g (or 0.25 g) SeaKem Gold (SKG) agarose into 250 ml screw-cap flask.
b. Add 50.0 ml (or 25.0 ml) TE Buffer; swirl gently to disperse agarose.
c. Loosen or remove cap, cover loosely with clear film, and microwave for 30 sec; mix gently and repeat for 10
sec intervals until agarose is completely dissolved.
d. Recap flask and return to 55-60ºC water bath and equilibrate the agarose in the water bath for 15 minutes or
until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: SeaKem Gold agarose works well for making PFGE plugs because it provides added strength to the plugs
minimizing breakage of plugs during the lysis and washing steps. The time and temperature needed to completely
dissolve the agarose is dependent on the specifications of the microwave used, and will have to be determined
empirically in each laboratory.

4. Label small transparent tubes (12 mm x 75 mm Falcon 2054 tubes or equivalent) with culture numbers.

5. Prepare Cell Suspension Buffer (100 mM Tris:100 mM EDTA, pH 8.0) as follows:

10 ml of 1 M Tris, pH 8.0
20 ml of 0.5 M EDTA, pH 8.0
Dilute to 100 ml with sterile Ultrapure water (CLRW)

6. Transfer ≈2 ml of Cell Suspension Buffer (CSB) to small labeled tubes. Use a sterile polyester-fiber or cotton
swab that has been moistened with sterile CSB to remove some of the growth from agar plate; suspend cells in CSB
by spinning swab gently so cells will be evenly dispersed and formation of aerosols is minimized.

Note: The minimum volume of the cell suspension needed will depend on size of the cuvettes or tubes used to measure
the cell concentration and are dependent on the manufacturer’s specifications for the spectrophotometer, turbidity meter,
or colorimeter. Keep suspensions on ice if you have more than 6 cultures to process or refrigerate cell suspensions if you
cannot adjust their concentration immediately.

7. Adjust concentration of cell suspensions to one of values given below by diluting with sterile CSB or by adding
additional cells.
a. Spectrophotometer: 610 nm wavelength, absorbance (Optical Density) of 1.00 (range of 0.8-1.0)
b. Dade Microscan Turbidity Meter: 0.40 - 0.45 (measured in Falcon 2054 tubes)
0.58 - 0.63 (measured in Falcon 2057 tubes)
c. bioMérieux Vitek colorimeter: ≈17-18% transmittance (measured in Falcon 2054 tubes)

Note: The values in Steps 7a, 7b and 7c give satisfactory results at CDC; each laboratory may need to establish the
optimal concentration needed for satisfactory results.

CASTING PLUGS

Label wells of PFGE plug molds with culture number. When reusable plug molds are used, put strip of tape on lower
part of reusable plug mold before labeling wells.

Note: Unused plug agarose can be kept at room temperature and reused 1-2 times. Microwave on low-medium power
for 10 -15 sec and mix; repeat for 5-10 sec intervals until agarose is completely melted. This agarose melts rapidly!

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 2 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

Note: Proteinase K solutions (20 mg/ml) are available commercially. Alternatively, a stock solution of Proteinase K can
be prepared from the powder in sterile Ultrapure water (CLRW). For best results, aliquot 300-500 μl into small tubes
and store in a freezer (-20ºC) until ready to use. Just before use, thaw appropriate number of vials needed for the
samples; keep Proteinase K solutions on ice. If the Proteinase K stock solution was prepared from powder, discard any
thawed solution at the end of the work day. Store commercially prepared Proteinase K solutions according to directions
provided by the supplier.

1. Transfer 400 μl (0.4 ml) adjusted cell suspensions to labeled, sterile 1.5 ml microcentrifuge tubes.

2. Add 20 μl of Proteinase K (20 mg/ml stock) to each tube and mix gently with pipet tip. (200 μl are needed for 10
cell suspensions.)

3. Add 400 μl melted 1% SeaKem Gold agarose to 400 μl cell suspension; mix by gently pipetting mixture up and
down a few times. Over-pipeting can cause DNA shearing. Maintain temperature of melted agarose by keeping
flask in beaker of warm water (55-60ºC).

4. Immediately, dispense part of mixture into appropriate well(s) of reusable plug mold. Do not allow bubbles to form.
Two plugs of each sample can be made from these amounts of cell suspension and agarose and are useful if repeat
testing is required. Allow plugs to solidify at room temperature for 10-15 min. They can also be placed in the
refrigerator (4ºC) for 5 minutes

Note: If disposable plug molds are used for making plugs with 1% SeaKem Gold agarose, use 200 μl cell suspension,
10 μl of Proteinase K (20 mg/ml stock) and 200 μl of agarose; up to 4 plugs can be made from these amounts of cell
suspension and agarose.

Note: The generation of cell suspension and the subsequent casting of the plugs should be performed as rapidly as
possible in order to minimize premature cell lysis. If large numbers of samples are being prepared, it is recommended
that they be processed in batches of ~10 samples at a time. Once the first batch of isolates are in the cell lysis incubation,
then start preparing the cells suspensions the next group samples, and so on. All batches can be lysed and washed
together, since additional lysis time will not affect the initial batches.

LYSIS OF CELLS IN AGAROSE PLUGS

Note: Two plugs (reusable molds) or up to four plugs (disposable molds) of the same strain can be lysed in the same
50ml tube.

1. Label 50 ml polypropylene screw-cap or 50 ml Oak Ridge tubes with culture numbers.

2. Prepare Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) as follows:
25 ml of 1 M Tris, pH 8.0
50 ml of 0.5 M EDTA, pH 8.0
50 ml of 10 % Sarcosyl (N-Lauroylsarcosine, Sodium salt) 2
Dilute to 500 ml with sterile Ultrapure water (CLRW)

2
The N-Lauroylsarcosine, Sodium salt can be added directly to the other ingredients and allowed to dissolve. See
page 12 of this document.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 3 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

3. Calculate the total volume of Cell Lysis/Proteinase K Buffer needed as follows:

a. 5 ml Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosyl) is needed per tube
(e. g., 5 ml x 10 tubes = 50 ml).
b. 25 μl Proteinase K stock solution (20 mg/ml) is needed per tube of the cell lysis buffer
(e. g., 25 μl x 10 tubes = 250 μl).
c. Prepare the master mix by measuring the correct volume of Cell Lysis Buffer and Proteinase K into appropriate
size test tube or flask and mix well.

Note: The final concentration of Proteinase K in the lysis buffer is 0.1 mg/ml and is different from the concentration that
was added to the cell suspension (0.5 mg/ml).

4. Add 5 ml of Proteinase K/Cell Lysis Buffer to each labeled 50 ml tube.

5. Trim excess agarose from top of plugs with scalpel, razor blade or similar instrument. Open reusable plug mold and
transfer plugs from mold with a 6-mm wide spatula to appropriately labeled tube. If disposable plug molds are used,
remove white tape from bottom of mold and push out plug(s) into appropriately labeled tube. Be sure plugs are
under buffer and not on side of tube.

Note: The excess agarose, plug mold, spatula, etc. are contaminated. Discard or disinfect appropriately.

6. Remove tape from reusable mold. Place both sections of the plug mold, spatulas, and scalpel in 90% ethanol, 1%
Lysol/Amphyll other suitable disinfectant. Soak them for 15 minutes before washing them. Discard disposable
plug molds or disinfect them in or 90% ethanol for 30-60 minutes if they will be washed and reused.

7. Place tubes in rack and incubate in a 54-55ºC shaker water bath or incubator for 1.5 - 2 h with constant and
vigorous agitation (150-175 rpm). If lysing in water bath, be sure water level is above level of lysis buffer in tubes.

8. Pre-heat enough sterile Ultrapure water (CLRW) to 54-55ºC so that plugs can be washed two times with 10-15 ml
water (200-250 ml for 10 tubes).

WASHING OF AGAROSE PLUGS AFTER CELL LYSIS

Note: Most laboratories will find that their plugs are sufficiently stable to perform the following washing steps at
54-55ºC. However, if you notice that your plugs are nicked along the edges or breaking it will be necessary for your
laboratory to lower the water bath or incubator to 50ºC for the following washing steps.

1. Remove tubes from water bath or incubator, and carefully pour off lysis buffer into an appropriate discard container;
plugs can be held in tubes with a screened cap or spatula.

Note: It is important to remove all of the liquid during this and subsequent wash steps by touching edge of tube or
screened cap on an absorbent paper towel.

2. Add at 10-15 ml sterile Ultrapure water (CLRW) that has been pre-heated to 54-55ºC to each tube and shake the
tubes in a 54-55ºC water bath or incubator for 10-15 min.

3. Pour off water from the plugs and repeat wash step with pre-heated water (Step 2) one more time.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 4 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

a. Pre-heat enough sterile TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) in a 54-55ºC water bath so that plugs
can be washed four times with 10-15 ml TE (400-600 ml for 10 tubes) after beginning last water wash.

4. Pour off water, add 10-15 ml pre-heated (54-55ºC) sterile TE Buffer, and shake the tubes in 54-55ºC water bath or
incubator for 10-15 min.

5. Pour off TE and repeat wash step with pre-heated TE three more times.

6. Decant last wash and add 5-10 ml sterile TE. Continue with step 1 in "Restriction Digestion" section or store plugs
in TE Buffer at 4ºC until needed. Plugs can be transferred to smaller tubes for long term storage.

Note: If restriction digestion is to be done the same day, complete Steps 1-3 of next section (Restriction Digestion)
during last TE wash step for optimal use of time.

RESTRICTION DIGESTION OF DNA IN AGAROSE PLUGS

Note: A small slice of the plug (not the entire plug) should be digested with the primary restriction enzyme because less
enzyme is required and other slices of the plug can be subjected to restriction analysis with secondary or tertiary
enzymes, according to the table below. The use of a secondary (or tertiary) enzyme is useful in situations where the
PFGE patterns obtained with the primary enzyme from two or more isolates are indistinguishable.

Primary Enzyme Secondary Enzyme

Organism
(Concentration) (Concentration)
XbaI SpeI
Cronobacter
(50 U/sample) (30 U/sample)

1. Label 1.5 ml microcentrifuge tubes with culture numbers; label 3 (10-well gel) or 4 (15-well gel) tubes for
Salmonella ser. Braenderup H9812 standards.
a. Pre-Restriction Incubation Step (highly recommended): Prepare a master mix by diluting the appropriate
10X restriction buffer (Roche Applied Science or equivalent) 1:10 with sterile Ultrapure water (CLRW)
according to the following table:

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory

180 μl 1800 μl 2700 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl
Total Volume 200 μl 2000 μl 3000 μl

b. Add 200 μl diluted restriction buffer (1X) to labeled 1.5 ml microcentrifuge tubes.
c. Carefully remove plug from TE with spatula and place in a sterile disposable Petri dish or on large glass slide.
d. Cut a 2.0 to 2.5 mm wide slice from each test samples and the appropriate number of S. ser. Braenderup H9812
standards with a single edge razor blade (or scalpel, cover slip, etc.) and transfer to tube containing diluted

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 5 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

restriction buffer. Be sure plug slice is under buffer. Replace rest of plug into the original tube that contains
5 ml TE buffer and store at 4ºC.

Note: PulseNet recommends that the combs with larger teeth (10 mm wide teeth) be used to cast the gels because
computer analysis of the gel lanes is more accurate and less tedious than analysis of gel lanes cast with combs with the
smaller teeth (5.5 mm). Using combs with smaller teeth is not advised. The number of slices that can be cut from the
plugs will depend on the skill and experience of the operator, integrity of the plug, and whether the slices are cut
vertically or horizontally (plugs made in disposable molds).

e. Incubate sample and control plug slices in a 37ºC water bath for 5-10 min or at room temp for 10-15 min.
f. After incubation, remove buffer from plug slice using a pipet fitted with 200-250 μl tip all the way to bottom of
tube and aspirate buffer. Be careful not to damage the plug slice with pipet tip and that plug slice is not
discarded with pipet tip.

2. Prepare the restriction enzyme master mix according to the following table3. May mix in the same tube that was
used for the diluted restriction buffer.

Note: Enzymes may be purchased in several different stock concentrations. The calculations outlined here are based on
using an enzyme at a concentration of 10 U/μl. If the enzyme used is of a different concentration, make necessary
adjustments to the volume of enzyme and water to achieve a final concentration of 50 U/ sample.

Note: Keep vial of restriction enzyme on ice or in insulated storage box (-20˚C) at all times.

Reagent μl/Plug Slice µl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory
174 μl 1740 μl 2610 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl
BSA (20mg/ml) 1 μl 10 μl 15 μl
Enzyme (10 U/μl) 5 μl 50 μl 75 μl

Total Volume 200 μl 2000 μl 3000 μl

Note: Addition of Bovine Serum Albumin (BSA; highly recommended): Several restriction enzyme vendors
specifically recommend the addition of 1X BSA to enzyme restriction mixtures while others do not. PulseNet
Central recommends adding BSA to all enzyme restriction mixtures to minimize the incidence of incomplete
restriction.

3. Add 200 μl restriction enzyme master mix to each tube. Close tube and mix by tapping gently; be sure plug slices
are under enzyme mixture.

4. Incubate sample and control plug slices in 37˚C water bath for 1.5-2 h.

5. If plug slices will be loaded into the wells (Option B, page 8), continue with Steps 1-4 of the next section
(CASTING AGAROSE GEL) approximately 1 h before restriction digest reaction is finished so the gel can
solidify for at least 30 minutes before loading the restricted PFGE plugs.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 6 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

CASTING AGAROSE GEL

A. Loading Restricted Plug Slices on the Comb:

1. Confirm that water bath is equilibrated to 55-60ºC.

2. Make volume of 0.5X Tris-Borate EDTA Buffer (TBE) that is needed for both the gel and electrophoresis running
buffer according to one of the following tables.

5X TBE:
Reagent Volume in milliliters (ml)
5X TBE 200 220
Clinical Laboratory Reagent
1800 1980
Water (CLRW)
Total Volume of 0.5X TBE 2000 2200

10X TBE:
Reagent Volume in milliliters (ml)
10X TBE 100 110
Clinical Laboratory Reagent
1900 2090
Water (CLRW)
Total Volume of 0.5X TBE 2000 2200

3. Make 1% SeaKem Gold (SKG) Agarose in 0.5X TBE as follows:

a. Weigh appropriate amount of SKG into 500 ml screw-cap flask.
b. Add appropriate amount of 0.5X TBE; swirl gently to disperse agarose.
i. Mix 1.0 g agarose with 100 ml 0.5X TBE for 14 cm wide gel form (10 wells)
ii. Mix 1.5 g agarose with 150 ml 0.5X TBE for 21 cm wide gel form (15 wells)
c. Loosen or remove cap and cover loosely with clear film, and microwave for 60 sec; mix gently and repeat for
15 sec intervals until agarose is completely dissolved.
d. Recap flask and return to 55-60ºC water bath and equilibrate the agarose in the water bath for 15 minutes or
until ready to use.

SAFETY WARNING: Use heat-resistant gloves when handling hot flasks after microwaving.

Note: Agarose LFTM (Amresco, X174) is the only acceptable alternative to SeaKem Gold, at this time. The time and
temperature needed to completely dissolve the agarose is dependent on the specifications of the microwave used and will
have to be determined empirically in each laboratory. Similarly, the optimal running time for each agarose will have to
be determined empirically in each laboratory.

4. A small volume (2-5 ml) of melted and cooled (55-60ºC) 1% SKG agarose may be wanted to seal wells after plugs
are loaded. Prepare as described above. Unused SKG agarose can be kept at room temperature, melted, and reused
several times.

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 7 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

Note: Place the gel form on a leveling table and adjust until perfectly leveled. Place the comb holder so the front part
(side with small metal screws) and teeth face the bottom of gel frame and the comb teeth touch the gel platform.

5. Remove restricted plug slices from 37ºC water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE.
Incubate at room temperature for 5 min.

6. Remove plug slices from tubes; put comb on bench top and load plug slices on the bottom of the comb teeth as
follows:
a. Load S. ser. Braenderup H9812 standards on teeth (lanes) 1, 5, 10 (10 well gel) or on teeth 1, 5, 10, 15 (15 well
gel).
b. Load samples on remaining teeth and note locations.

7. Remove excess buffer with tissue or kimwipe. Allow plug slices to air dry on the comb for 5-10 minutes or seal
them to the comb with 1% SKG agarose (55-60ºC).

8. Position comb in leveled gel form and confirm that the plugs slices are correctly aligned on the bottom of the comb
teeth, and that the lower edge of the plug slice is flush against the black platform.

9. Carefully pour the agarose (cooled to 55-60ºC) into the gel form and remove any bubbles or debris.

10. Put black gel frame in electrophoresis chamber. Add 2-2.2 L freshly prepared 0.5X TBE. Close cover of unit. The
amount of buffer needed depends on whether residual buffer was left in tubing or if unit was flushed with water
after the last gel was run.

11. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute (setting of ≈70) and cooling module (14ºC)
approximately 30 minutes before gel is to be run.

12. Remove comb after gel solidifies, about 30-45 minutes.

13. Fill in wells of gel with melted and cooled (55-60ºC) 1% SKG Agarose (optional). Unscrew and remove end gates
from gel form; remove excess agarose from sides and bottom of casting platform with a tissue or kimwipe. Keep
gel on casting platform and carefully place gel inside black gel frame in electrophoresis chamber. Close cover of
chamber.

B. Loading Restricted Plug Slices into the Wells:

1. Follow steps 1-4 in Section A on pages 7-8 (Loading Restricted Plug Slices on the Comb).

Note: Place the gel form on a leveling table and adjust until perfectly leveled before pouring gel. Position the comb
holder so that front part (side with small metal screws) and teeth face the bottom of gel and the bottom edge of the
comb is 2 mm above the surface of the gel platform.

2. Cool melted SKG agarose in 55-60ºC water bath for 15-20 min; carefully pour agarose into gel form (casting stand)
fitted with comb. Be sure there are no bubbles.

3. Put black gel frame in electrophoresis chamber. Add 2-2.2 L freshly prepared 0.5X TBE. Close cover of unit. (The
amount of buffer depends on whether residual buffer was left in tubing, or if unit was flushed with water after the
last gel was run).
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 8 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

4. Turn on power supply, pump calibrated to a flow rate of 1 liter/minute (setting at ~70), and cooling module (14ºC)
approximately 30 minutes before gel is to be run.

5. Remove restricted plug slices from 37ºC water bath. Remove enzyme/buffer mixture and add 200 μl 0.5X TBE.
Incubate at room temperature for 5 minutes.

6. Remove comb after gel solidifies, about 30 – 45 minutes.

7. Remove restricted plug slices from tubes with tapered end of spatula and load into appropriate wells. Gently push
plugs to bottom and front of wells with wide end of spatula. Manipulate position with spatula and be sure that are
no bubbles.
a. Load S. ser. Braenderup H9812 standards in wells (lanes) 1, 5, 10 (10 well gel) or in wells 1, 5, 10, 15 (15 well
gel).
b. Load samples in remaining wells.

Note: Loading the plug slices can be tedious; each person has to develop his/her own technique for consistently
placing the plug slices in the wells so the lanes will be straight and the bands sharp.

8. Fill in wells of gel with melted 1% SKG Agarose (equilibrated to 55-60ºC). Allow to harden for 3-5 min. Unscrew
and remove end gates from gel form; remove excess agarose from sides and bottom of casting platform with a tissue
or kimwipe. Keep gel on casting platform and carefully place gel inside black gel frame in electrophoresis chamber.
Close cover of chamber.

ELECTROPHORESIS CONDITIONS

1. Select following conditions for Cronobacter strains restricted with XbaI or SpeI:
a. Select following conditions on CHEF Mapper
Auto Algorithm
25 kb - low MW
286 kb - high MW
Select default values except where noted by pressing "enter."
Change run time to 18 - 19 h (See note below)
(Default values: Initial switch time = 1.8 s; Final switch time = 25 s)

b. Select following conditions on CHEF-DR III

Initial switch time: 1.8 s
Final switch time: 25 s
Voltage: 6 V
Included Angle: 120°
Run time: 18-19 h (See note below)

c. Select following conditions on CHEF-DR II

Initial A time: 1.8 s
Final A time: 25 s
Start ratio: 1.0 (if applicable)
Voltage: 200 V
Run time: 19-20 h (See note below)

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 9 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

Note: The electrophoresis running times recommended above are based on the equipment and reagents used at the CDC.
Run times may be different in your laboratory and will have to be optimized for your gels so that the lowest band
in the S. ser. Braenderup H9812 standard migrates 1.0-1.5 cm from the bottom of the gel.

Note: Make note of the initial milliamp (mAmp) reading on the instrument. The initial mAmps should be between 110-
150 mAmps. A reading outside of this range may indicate that the 0.5X TBE buffer was prepared improperly and the
buffer should be remade.

Day 2

STAINING AND DOCUMENTATION OF PFGE AGAROSE GEL

Note: The following staining procedure describes the use of ethidium bromide to stain PFGE gels. Alternate DNA
stains may be used. Please see the “Alternate DNA Stains-Results and Recommendations” posting within the PulseNet
Documents forum on the SharePoint site for additional information.

1. When electrophoresis run is over, turn off equipment; remove and stain gel with ethidium bromide by diluting 40 μl
of ethidium bromide stock solution (10 mg/ml) with 400 ml of Ultrapure water (CLRW). This volume is for a
staining box that is approximately 14 cm x 24 cm; a larger container may require a larger amount of staining
solution. Stain gel for 20-30 min in covered container.

Note: Ethidium bromide is toxic and a mutagen. Stock solutions of 10 mg/ml Ethidium Bromide (EtBr) in water are
available from several commercial companies (Amresco X328; Bio-Rad, 161-0433; Sigma, E-1510). The diluted
solution can be kept in dark bottle and reused 6-8 times before discarding according to your institution's guidelines for
hazardous waste. CDC does not recommend disposing of EtBr down the drain. Aqueous solutions containing EtBr
can be filtered through charcoal or degraded using activated carbon destaining or “tea” bags from Amresco (E732-
25 Destaining Bags) or other companies, which effectively and safely remove EtBr from solutions and gels. Once
the EtBr is removed, the treated aqueous solutions can be discarded down the drain. If you have further questions
about EtBr please refer to the Material Safety Data Sheets (MSDS) provided by the vendor or manufacturer.

Note: Currently, the only acceptable alternative stain options are GelRedTM (Biotium, 31010), SYBR® Safe
(Invitrogen, S-33102) and SYBR® Gold (Invitrogen, S-11494). Labs are strongly encouraged to follow
manufacturer’s instructions and test stains in their labs before adopting them for routine use. If one of the
alternative stains is used, the destaining steps should be omitted.

2. Destain gel in approximately 500 ml CLRW for 60 - 90 min, changing water every 20 minutes. Capture image using
a Gel Doc 1000, 2000, EQ, XR, or equivalent documentation system. If too much background is observed destain
for an additional 30-60 min.

3. Follow directions given with the imaging equipment to save gel image as a *.1sc file; convert this file to *.tif file for
analysis with the BioNumerics software program. The gel image should fill the entire window of the imaging
equipment (computer) screen (without cutting off wells or lower bands). Ensure that the image is in focus and
that there is little to no saturation (over-exposure) in the bands (signified by red pixilation in the QuantityOne or
ImageLab software). Additional instructions are provided in PNL07 of the PulseNet QA/QC manual.

4. Drain buffer from electrophoresis chamber and discard. Rinse chamber with 2 L Ultrapure water (CLRW) or, if
unit is not going to be used for several days, flush lines with water by letting pump run for 5-10 min before draining
water from chamber and tubing.
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 10 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

5. If the lowest band in the H9812 standard does not migrate within 1-1.5 cm of the bottom of the gel, the proper run
time will need to be determined empirically for the conditions in each laboratory.

Note: The following options are available if PFGE results do not have to be available within 24-28 hours:

− Plugs can be lysed for longer periods of time (3-16 hours).

− The washing steps with TE to remove the lysis buffer from the PFGE plugs can be done for longer periods
of time (30-45 min) and at lower temperatures (37°C or room temperature). They can be started on Day 1
and finished on Day 2 after overnight refrigeration of the plugs in TE.

Use of trade names and commercial sources is for identification purposes only and does not imply endorsement by CDC
or the U.S. Department of Health and Human Services.

NOTE: CLIA LABORATORY PROCEDURE MANUAL REQUIREMENTS

Efforts have been made to assure that the procedures described in this protocol have been written in accordance with
the 1988 Clinical Laboratory Improvement Amendments (CLIA) requirements for a procedure manual (42 CFR
493.1211). However, due to the format required for training, the procedures will require some modifications and
additions to customize them for your particular laboratory operation.
Any questions regarding the CLIA requirements for a procedure manual, quality control, quality assurance, etc.,
should be directed to the agency or accreditation organization responsible for performing your laboratory's CLIA
inspection. In addition, some states and accreditation organizations may have more stringent requirements that will need
to be addressed.

Formulas of Selected Reagents used in PulseNet Standardized Laboratory Protocol for PFGE

Tris:EDTA Buffer, pH 8.0 (TE, 10 mM Tris:1 mM EDTA, pH 8.0)

10 ml of 1 M Tris, pH 8.0
2 ml of 0.5 M EDTA, pH 8.0
Dilute to 1000 ml with sterile Ultrapure water (CLRW)

Cell Lysis Buffer (50 mM Tris:50 mM EDTA, pH 8.0 + 1% Sarcosine + 0.1 mg/ml Proteinase K)
25 ml (50 ml) of 1 M Tris, pH 8.0
50 ml (100 ml) of 0.5 M EDTA, pH 8.0
50 ml (100 ml) 10% N-Lauroylsarcosine, Sodium salt (Sarcosyl)
OR
5 g (10 g) of N-Lauroylsarcosine, Sodium salt (Sarcosyl) 3
Dilute to 500 ml (1000 ml) with Sterile Ultrapure water (CLRW)

Add 25 μl Proteinase K stock solution (20 mg/ml) per 5 ml of cell lysis buffer just before use for a final
concentration in the lysis buffer of 0.1 mg/ml Proteinase K.

3
If Sarcosyl powder is added directly to the other components of this reagent, warm the solution to 50-60ºC for 30-
60 minutes, or leave at room temperature for ≈2 hours to completely dissolve the Sarcosyl; adjust to the final
volume with sterile Ultrapure water (CLRW).
AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 11 of 12
STANDARD OPERATING PROCEDURE FOR PULSENET PFGE OF CRONOBACTER CODE: PNL31
Effective Date:
SPECIES 12 16 2013

Use the following calculations for SpeI (30 Units/plug slice):

Reagent μl/Plug Slice μl/10 Plug Slices μl/15 Plug Slices

Sterile Clinical Laboratory
178.25 μl 1782.5 μl 2673.75 μl
Reagent Water (CLRW)
10X Restriction Buffer 20 μl 200 μl 300 μl
BSA (20mg/ml) 1 μl 10 μl 15 μl
SpeI (40 U/μl) 0.75 μl 7.5 μl 11.25 μl

Total Volume 200 μl 2000 μl 3000 μl

Note: Keep vial of restriction enzyme on ice or in insulated storage box (-20ºC) at all times.

5. FLOW CHART:

6. BIBLIOGRAPHY:
1. Brengi, et al. 2012. Development and validation of a PulseNet standardized protocol for subtyping Isolates
of Cronobacter species. Foodborne Pathog Dis. 9:861 – 867.
2. Ribot, at al. 2006. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of
Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis. 3:59 – 67.

7. CONTACTS:

8. AMENDMENTS:

AUTHORED BY: REVIEWED BY: AUTHORIZED BY:

Page 12 of 12
 STANDARD OPERATING PROCEDURE FOR COMPUTER SUPPLIES AND CODE: PND01
Effective Date:
EQUIPMENT 2 19 2008

1. PURPOSE: To describe the minimal requirements for computer hardware and software needed for analysis of
PulseNet PFGE gels.

2. SCOPE: This procedure applies to all PulseNet participating laboratories analyzing PFGE gels.

3. DEFINITIONS/TERMS:

3.1 PFGE: Pulsed-field Gel Electrophoresis

3.2 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
3.3 CDC: Centers for Disease Control and Prevention

4. RESPONSIBILITIES/PROCEDURE:

4.1 Participating laboratories must use the following software and hardware
4.1.1 BioNumerics Software v3.0, v3.5, v4.01, or v5.0
4.1.2 CDC PulseNet masterscripts
4.1.3 Personal computer equipped with an Intel Pentium CPU or better, 256 MB RAM or more, 65 K
color graphics or better and Windows 98, Windows NT 4.0, or higher
4.1.4 Keyboard, mouse, CD-ROM
4.1.5 High resolution screen 1024x768 or higher; true color
4.1.6 Large screen recommended (17" or more)

4.2 For quotes and additional information in USA and Canada, contact Applied Maths, Inc.
4.3 For quotes and additional information in Europe, contact Applied Maths BVBA.

Use of trade names and commercial sources is for identification only and does not imply endorsement by CDC or
the U.S. Department of Health and Human Services.
5. FLOW CHART:
6. BIBLIOGRAPHY:
7. CONTACTS:
7.1 Applied Maths, Inc.

512 East 11th Street, Suite 207

Austin, TX 78701
Phone: 512-482-9700
Fax: 512-482-9708
E-mail: [email protected]
www.applied-maths.com

7.2 Applied Maths BVBA

Keistraat120
B-9830 Sint-Martens-Latem
Belgium
Phone: +32-9-2222-100
Fax: +32-9-2222-102
E-mail: [email protected]
www.applied-maths.com

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 1
STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS CODE: PND02
Installation and Use of BioNumerics Gel Analysis Software for PulseNet Effective Date:
Participants 5 9 05

1. PURPOSE : To describe the guidelines for standardization of TIFF image analysis in order to accurately
compare PFGE patterns between PulseNet participating laboratories.

2. SCOPE: This procedure applies to all analyses submitted to PulseNet, thereby allowing comparison of results
with other PulseNet laboratories.

3. DEFINITIONS/TERMS:

3.1 SOP: Standard Operating Procedure

3.2 PFGE: Pulsed-field Gel Electrophoresis
3.3 TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
3.4 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium

4. RESPONSIBILITIES:

4.1 After an electronic image of a gel is obtained using the procedure in SOP PNL07, the file MUST be
analyzed using the PulseNet customized version of BioNumerics Analysis Software version 3.0 or higher.

4.2 The SOPs PNG05, PNL01 – PNL06 and PND01 contain settings and instructions to allow users to create
and compare analyzed PFGE fingerprint patterns sent to the PulseNet national databases. Masterscripts for
setting up databases in E. coli, Listeria, Salmonella, Shigella, and Campylobacter are available from the
PulseNet Task Force by sending an e-mail to [email protected]. In order for results to be compatible with the
PulseNet national databases, databases must be set up using the masterscripts provided by the PulseNet
Task Force.

4.3 TIFFs can either be copied to the appropriate images directory or imported directly into BioNumerics.

4.4 Local labs can add information fields to their databases; however, they should add their lab ID either to the
beginning or end of the information field name. This is recommended to ensure that the local labs are able
to differentiate between CDC fields and their local lab fields.

4.5 When creating a bundle file to be shared with other PulseNet participating labs, and/or on the WebBoard,
the “PulseNet Bundle” tool must be used.

5. PROCEDURE:

5.1 Installing BioNumerics Software

5.1.1 Insert the BioNumerics CD into the CD-ROM drive and click on “Install BioNumerics.”
5.1.2 Note the directory the installation will be in and use the default setting to install a sample
database (DemoBase).
5.1.3 Click “Yes” to create a shortcut icon
5.1.4 Click “OK” for the protection key installer
5.1.5 Click “Next” to install the sentinel system driver
5.1.6 Accept the terms and click “Next”
5.1.7 Choose the default folder and then click “Next”
5.1.8 Choose the default “Complete” setup type and then click “Next”
5.1.9 Clink “Install” to begin the installation process
5.1.10 Insert the license string, which is found in the BioNumerics package
5.1.11 Click “Finish”

5.2 Creating a new database

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 7
STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS CODE: PND02
Installation and Use of BioNumerics Gel Analysis Software for PulseNet Effective Date:
Participants 5 9 05

5.2.1 Double-click on the BioNumerics icon and click the “New” button.
5.2.2 When the new database window appears, enter a name for the new database and click
“Next.”
5.2.3 Choose which directory you would like to install the database into. Click “Next” to accept the
default directory or change the directory path by using the browse key, and then click “Next.”
5.2.4 When asked if you would like to create log files for the new database, select “Yes” and click the
“Finish” button. The log files allow you to track any changes made to the database.

5.3 Running masterscripts

5.3.1 Insert the most recent PulseNet masterscripts CD (provided by PulseNet) into the CD-ROM drive.
5.3.2 Start the BioNumerics software, highlight the database name that you wish to customize for
PulseNet and click the “Analyze” button.
5.3.3 In the upper toolbar click “Scripts” and select “Run script from file…”
5.3.4 Choose “Install.BNS” from the PulseNet Master Scripts CD.
5.3.5 Select the appropriate organism and enter your lab ID; then click “OK.”
5.3.6 The program will ask to be restarted, click “OK” to finish the installation of Master Scripts.

5.4 Changing database settings

5.4.1 Highlight the database of interest and click “Settings.”
5.4.2 By clicking on the tabs, various settings can be changed, including the background color and the
order of the database fields.

5.5 Importing a TIFF into BioNumerics

5.5.1 Highlight the database of interest and click “Analyze.”
5.5.2 Right-click under the “Files” panel in the main BioNumerics screen and click on “Add new
experiment file…,” or click on the folder shortcut button at the top of the Files menu.
5.5.3 “Browse,” and choose the location of the TIFF, highlight it, and click “Open.”
5.5.4 In BioNumerics v4.0, a confirmation window will ask, “Do you want to edit the image before
adding it to the database?”
5.5.4.1 Choose “No” if you do not need to edit the image.
5.5.4.2 If you choose “Yes,” you can rotate or crop the image, or invert colors.
5.5.5 There should now be a red “N” in front of the TIFF name (the “N” denotes a new TIFF that has
not been analyzed).

5.6 Saving a TIFF into an organism-specific BioNumerics database from an e-mail or WebBoard
5.6.1 When you save a TIFF to analyze in BioNumerics, you will need to save it in a specific file
location.
5.6.2 Save the TIFF in the “Images” folder in the BioNumerics directory for the specified organism. For
example, to save a TIFF in the E. coli BioNumerics database, you would save it under:
C:\Program Files\BioNumerics\data\Ecoli\Images.
5.6.3 The next time the specified database is open, the TIFF will appear under the “Files” panel with a
red “N” in front of it.
5.6.4 Note: The TIFF will not appear if it is saved in the “Images” folder while BioNumerics is open.
To see the newly imported TIFF, close, then reopen BioNumerics.

5.7 Analyzing a gel

5.7.1 Locate the TIFF you wish to analyze in the “Files” area and double-click on the file name, or
highlight the TIFF name and click on the blue arrow shortcut button.
5.7.2 When the “Fingerprint File” window opens, click on the “Edit fingerprint data” button.
5.7.3 Select the appropriate fingerprint type (this will be the enzyme used for the majority of the gel)
and click “OK.”

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 7
STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS CODE: PND02
Installation and Use of BioNumerics Gel Analysis Software for PulseNet Effective Date:
Participants 5 9 05

5.7.4 The gel should be white with black bands. If the gel appears black with white bands, go to “Edit
settings” and check the box for “Inverted values.”
5.7.5 Once the Fingerprint data window opens, the selected TIFF image appears in a green box.
5.7.6 Using the green nodes, adjust the size of the green box so that the left and right sides of the green
box are at the edge of the left and rightmost gel lanes. Adjust the top of the green box so that it is
just below the wells of the gel. Position the bottom of the green box at the bottom of the gel or the
bottom of the TIFF (if the bottom of the gel is not visible).
5.7.7 If the gel is curved, holding down the <Shift> key while adjusting the nodes will allow for the box
to curve around the gel.
5.7.8 Next, indicate the lanes on the gel by clicking on the “Auto search lanes” button (magnifying
glass) to perform a lane search.
5.7.9 The Search lanes window will appear and prompt you for an estimated number of lanes on the gel.
This number doesn’t have to be exact but it should be close to the number of lanes since this will
help the program to designate the correct number of lanes. Enter the number of lanes and click
“OK.” Each lane that encompasses the DNA fingerprint is termed a “Gel Strip.”
5.7.10 If the exact number of lanes is known, you may choose to add them by choosing “Lanes” and
“Define group of lanes…” and entering the exact number of lanes on the gel.
5.7.11 When the lane auto search is complete, the “Gel Strip” may need to be adjusted to encompass the
entire PFGE pattern. Blue nodes located inside of the “Gel Strips” allow these lane indicators to be
moved and curved to match the lanes on the gel. To move the entire gel strip, click on any node
and move the gel strip left or right. To adjust the curvature of a gel strip, hold down the <Shift>
key and click on a node and drag it (hold down the mouse button while you move it) to curve the
gel strip. How the gel strip is curved depends on the selected node.
5.7.12 Further adjustments to the fingerprint conversion can be made in the Fingerprint conversion
settings window. Click on the “Edit Settings” button or choose “Edit” then “Edit Settings” from
the top menu to open this window. From this window you can adjust the thickness of the image
gel strips so that they fit the gel lanes. You can also adjust the number of nodes in the lane
indicators to allow for more adjustment flexibility. When you are done with adjustments click
“OK” to close the window and apply the changes.
5.7.13 Once the gel strips have been placed and adjusted to your satisfaction, adjustments to the
appearance of the gel image can be made in the “Gel tone curve” window. To open the window,
click on the “Edit” menu and click on “Edit tone curve.” The “Gel tone curve” window has a set of
image adjustment buttons and a pair of gel lane windows that show a “before” and “after” preview
of any adjustments before they are applied. Furthermore, by clicking in the bottom preview
window and dragging the mouse, the gel image can be moved around within the “after” window.
5.7.14 The “Linear” adjustment on the “Gel tone curve” window is a good starting point to adjust the
color of a gel image. This adjustment selects for the “best fit” Optical Density (OD) range or
densitometric curve values for optimizing the TIFF image. To use this function click on the
“Linear” button. Other adjustments that can be used to improve a gel image are “Enhance weak
bands” and “Enhance dark bands.” These two functions are complementary, so using one and then
the other will cancel any effect. Click on the “Enhance weak bands” button to apply this
adjustment. The button can be clicked a number of times to increase the level of adjustment.
5.7.15 When satisfied with all tone curve adjustments click “OK” to accept the changes (or click
“Cancel” to reject any changes), which will close the “Gel tone curve” window.
5.7.16 When you are satisfied with the gel strip positions and any image adjustments that have been
made, click on the “Next step” button (right arrow) to proceed to Step 2 of Analysis.
5.7.17 The goal of Step 2 is to place the Curve strips so that they are located in the area of the lane that
gives the most accurate representation of the gel bands. The densitometric curve for the selected
lane is shown on the right side of the window. The curve peaks indicate where the gel lane is
“darkest” or has the highest optical density. Click on the nodes in the Curve strips and drag the
mouse to move the whole strip from left to right. By holding down the <Shift> key and clicking

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 7
STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS CODE: PND02
Installation and Use of BioNumerics Gel Analysis Software for PulseNet Effective Date:
Participants 5 9 05

on a node, the Curve strip can be curved. You should avoid placing the curve strip in areas with
artifacts (specks, etc.) on the gel.
5.7.18 Once the Curve strips have been successfully placed, click on the “Next Step” button to go to Step
3 of Analysis, which is a step for Normalization where the gel lanes are normalized against the
PulseNet global standard.
5.7.19 Note: During any step of analysis, you can save your work, undo and redo actions, and zoom in or
out by clicking on the various buttons in the upper toolbar.
5.7.20 Step 3, “Normalization,” allows laboratories to compare PFGE results and is the most important
step of analysis. Click on a reference lane to select it (the number above the lane should turn
yellow) and click on the “Use selected lane as reference lane” button (looks like a weight). This
will mark the lane as a reference lane. Repeat this process for all of the reference lanes.
5.7.21 Match the bands in the reference lanes from the gel strips with the global standard band markers
(flags with fragment sizes on them).
5.7.22 To mark a gel strip reference lane band, first select the desired lane by clicking on it. The gel strip
number turns yellow when selected. Place the band selection cursor (small black arrow) on a band
of a reference lane by clicking on it with the mouse and then clicking on its corresponding global
standard lane band marker (the selected band marker will turn white). You can also drag the
mouse within the gel strip (hold down the <Tab> key to take off “Snap to Peaks” and move pixel
by pixel).
5.7.23 Note: Make sure to click the band first and then the global standard band marker to ensure the
correct band marker is highlighted. Enlarging the image using the zoom buttons helps with
placement of band markings.
5.7.24 Once both of the markers are correctly placed, press the <Enter> key to insert the normalization
marker. If you make a mistake in placing the normalization marker, then you can press the
<Delete> key to erase it. Repeat these steps to mark the rest of the bands in the reference lanes.
5.7.25 Another option is to mark only some of the bands for each reference lane. Click on the “Auto
assign reference positions” button to have BioNumerics search and place the normalization
markers. When the “Confirmation” window appears stating, “Do you want to preserve the existing
bands?” click “Yes” to save the selected bands that have been marked. Double-check to make sure
the correct bands in the reference lanes of the gel were matched up with the correct band markers.
5.7.26 After the bands for the reference lanes have been marked, and their placements are correct, then
click on the “Show normalized view” button to show the gel strips as they will appear when
normalized. If changes are made in the normalized view, click on the “Update normalization”
button to update normalization.
5.7.27 To check normalization, click on “Normalization” and “Show Distortion Bars” (in version 3.5,
this option is saved). The gel strip lanes will now show colored distortion bars to the right of the
gel lanes. The colors in the distortion bars indicate the degree of adjustment that was made to each
section of the gel lane. The colors run from light blue for a small adjustment to red and black for a
large adjustment. An unusually high amount of orange, red or black in the distortion bar may
indicate a normalization error, and should be examined. The colors that are seen should be
consistent as they move horizontally across the gel lanes. In the case of an abnormal gel run, dark
distortion bars may remain, even if normalization was performed correctly.
5.7.28 Once the normalized gel strips have been checked for any normalization errors, click on the “Next
step” button to proceed to Step 4 to complete band marking.
5.7.29 Click on the “Auto search bands” button from the toolbar to have BioNumerics search the lanes
for band positions. When the “Band search” window appears, accept the default search values and
click “OK” to perform the band auto search. If the gel is atypical, adjust the minimum profiling
percentage. When the “Confirmation” window appears with a question that asks, “There are
already some bands defined on the gel. Do you want to keep the existing bands?” click “Yes” to
preserve the existing bands marked, “No” for BioNumerics to auto search for all bands again, or
“Cancel.”

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 7
STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS CODE: PND02
Installation and Use of BioNumerics Gel Analysis Software for PulseNet Effective Date:
Participants 5 9 05

5.7.30 After bands are found by the auto search function, look at the whole gel and delete erroneous or
unnecessary bands. Be sure to go through each lane individually and check band markings by
looking at a printout of the gel image. This will ensure that extra bands, such as those near the top
or bottom of a TIFF image, are deleted. If artifacts are present on a TIFF image or the TIFF image
is dark, the auto search function may find many extra bands.
5.7.31 Refer to the document “PulseNet Gel Analysis Guidelines” (PND04) for more information on how
to mark bands.
5.7.32 To manually insert a band mark, click on the position where you would like the band, and this will
put the band placement cursor where the band mark will be placed. Press the <Enter> key to insert
the band mark. To remove a band mark, highlight the band mark and press the <Delete> key. If
you are uncertain about a band mark, then you can classify a band mark as uncertain (press <F5>),
and this mark will not be included in comparisons (dendrograms, etc.) unless you choose to have
them included. Do not place more than two uncertain bands per gel. If you need more, you should
rerun the corresponding isolates or the entire gel. To access bands that may be marked outside of
the standard, you can un-normalize the gel.
5.7.33 Once all of the lanes have been checked, click the “Save” button to save the normalized gel strips
with band marks. Close the Fingerprint data window after saving. This completes the analysis.

5.8 Changing enzymes/Fingerprint type

5.8.1 It is now time to link lanes; but first, make sure the correct fingerprint type (e.g., PFGE-XbaI,
PFGE-BlnI, etc.) is indicated. If the lanes are linked with incorrect fingerprint types, possible
duplicate entries in the database can occur.
5.8.2 To change the fingerprint type, right-click on the desired lane and select “Change fingerprint type
of lane,” then select the appropriate enzyme.

5.9 Linking isolates to patterns/database entries

5.9.1 To link a lane, either right-click on the desired lane and select the “Link lane” option or highlight
the lane and click on the pink arrow on the top toolbar.
5.9.2 The “Link lane” window will appear and is where a unique isolate number or identifier should be
entered. This code will be known as a “Key” in BioNumerics. Click “OK” after entering the new
key. If the key is not currently present in the database, then a “Confirmation” window appears to
ask if you would like to create the new key. If the key is correct, then click “Yes.” If the key is
incorrect, click “Cancel” and reenter the key to make sure the key is correct.
5.9.3 If an isolate is a re-run, a different “Confirmation” window appears to ask if you would like to
create a duplicate key. If you would, click “Yes.” This will now create the isolate with a “/#1” at
the end; all the demographic information will stay the same.
5.9.4 Do not upload isolates with a “/#” at the end of them. Pull up all versions of the pattern and choose
which one looks the best and should be included in the national database. Double-click on the
isolate and click on the “Edit Database Fields” icon (lightning bolt with green “i”). In the
“Patterns” box, click on the button that corresponds to the “best pattern.” In the PFGE fingerprint
box that opens, click on the “Make first pattern…” button (this button will not appear if the pattern
you have chosen is already the first pattern). If the duplicate that was just linked is the best pattern,
it will no longer have a “/#” behind it and can be uploaded.
5.9.5 Do not link or upload standard/reference lanes. The only exception to this rule is for submission of
certification results.
5.9.6 To enter isolate information for a gel lane, double-click on the desired lane in the “Fingerprint
file” window. This will open the “Entry edit” window. Click on the “Edit database fields” (green
“i”) button to open the customized PulseNet “Entry properties” window. Note that data can be
entered in the “Entry edit” window; however, the data will be lost if you then open the “Entry
Properties” window without first clicking “OK.”
5.9.7 Enter the isolate information into data fields in the customized PulseNet “Entry properties”
window. Most of the data fields use drop-down menus (pick lists) containing fixed values, as well

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 7
STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS CODE: PND02
Installation and Use of BioNumerics Gel Analysis Software for PulseNet Effective Date:
Participants 5 9 05

as previous entries that were used, however, data can also be typed into these fields. In order to
enter correct serotype, use only the drop-down menu to enter serotype or antigenic formula
information for any specific isolate.
5.9.8 Antibiogram types can be entered for an isolate by clicking on the “Antibiotics” button to open the
isolate antibiotic screen. Set an antibiotic resistance level using the pick lists. To add an antibiotic
to the list click “Add,” and enter the new antibiotic name into the text box and click “OK.”
However, only the antimicrobials on the original list will be uploaded. Click “OK” when done.
5.9.9 Repeat entering information for all isolates on the gel.

5.10 Uploading data

5.10.1 Connecting to the PulseNet server is a two-part process. First, authenticate to the CDC firewall
using a SecurID key fob issued by PulseNet. You must pass analysis certification for each
organism database in order to have access to each database. If you are certified for analysis, but do
not have a SecurID, contact the PulseNet Database Team at the CDC. Next, connect to the server
using the BioNumerics software.
5.10.2 After connecting to the server, open the gel with the isolates that need to be uploaded. Click
“Submit fingerprint file to server” (lightning bolt with a blue arrow). In the “Submit fingerprint”
window, select only the lanes that need to be uploaded. Check or uncheck “Submit antibiotics
data” and “Submit biochemical data” as necessary. Then click “OK” to upload the isolates to the
PulseNet database.
5.10.3 If you need to resubmit gel information (i.e., you need to update a serotype or demographic
information), make sure to select all lanes that were previously submitted to the database.
Otherwise, those lanes not selected will become unlinked in the national database.

5.11 Creating comparisons and performing cluster analyses

5.11.1 Select the isolates that you would like to include in your comparison:
5.11.1.1 <Ctrl> + left click; <Shift> + left click; or left click + <space bar>
5.11.2 Or you can select isolates by querying the local database:
5.11.2.1 Several queries can be performed using the script icons on the main
BioNumerics screen.
5.11.2.2 There are queries for: isolate information, pattern information, antibiotics
resistance, and biochemical information.
5.11.2.3 The binoculars can be used to search for information in any field.
5.11.2.4 Use a wildcard (*) around the search criteria to ensure that all entries with the
search string are found.
5.11.3 In the upper toolbar click “Comparison” and select “Create new comparison.”
5.11.4 When the comparison screen opens, highlight the enzyme you wish to show by clicking on the
picture of the gel located next to the enzyme in the bottom toolbar.
5.11.5 In the upper toolbar click on “Clustering” and select “Calculate,” then choose “Cluster analysis
(similarity matrix)…”
5.11.6 In the Comparison settings window the Similarity coefficient should be Band based using the Dice
coefficient, and the Dendrogram type should be set to UPGMA.
5.11.7 You may have to adjust the Position tolerance settings for your dendrogram. You can do so by
clicking on the “Position tolerances…” button; however, PulseNet suggests using an Optimization
of 1.50 and a Position tolerance of 1.50. Click “OK” to close the Position tolerance settings
window.
5.11.8 Click “OK” in the Comparison settings window to create the dendrogram tree structure in the
dendrogram area of the Comparison window.
5.11.9 To compare two isolates, select the entries and click “Comparison” in the upper toolbar. Select
“Compare two entries” and highlight the enzyme that you wish to show.

6. FLOW CHART:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 7
STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS CODE: PND02
Installation and Use of BioNumerics Gel Analysis Software for PulseNet Effective Date:
Participants 5 9 05

7. BIBLIOGRAPHY:

Weinberg, Sandy. GOOD LABORATORY PRACTICE REGULATIONS. Second edition. Marcel Dekker, Inc.
USA (1995).

8. CONTACTS:

8.1 PulseNet Database Team

(404) 639-4558
[email protected]

9. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 7
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

1. PURPOSE: To explain the procedure for obtaining access to and using the PulseNet/OutbreakNet
SharePoint site.

2. SCOPE: This procedure applies to all PulseNet participants who need or have access to the
PulseNet/OutbreakNet SharePoint site. OutbreakNet participants should contact the CDC Outbreak
Response Team [email protected] to obtain access to SharePoint and/or with any
questions about OutbreakNet’s terms of use.

3. DEFINITIONS/TERMS:

3.1 PulseNet/OutbreakNet SharePoint site: A closed, web-based discussion forum used for
communication among PulseNet/OutbreakNet participants. The PulseNet/OutbreakNet
SharePoint site is open to all laboratory staff at PulseNet participating laboratories and
OutbreakNet Epidemiologists working in collaboration with these laboratories. When
approved by PulseNet, U.S. food regulatory staff and PulseNet International representatives
may also have access. This SharePoint site is not open to university or private industry
personnel unless their inclusion is deemed to be in the interest of public health. Throughout
this document, the PulseNet/OutbreakNet SharePoint site will be referred to as “SharePoint.”
(http://partner.cdc.gov/sites/NCEZID/DFWED/EDLB/PulseNet)
3.2 SharePoint: SharePoint is a web-based tool developed by Microsoft that can be used for many
different functions. PulseNet/OutbreakNet use it for document sharing, announcements, a
calendar, website links and discussion forums.
3.3 PFGE: Pulsed-field Gel Electrophoresis
3.4 CDC: Centers for Disease Control and Prevention
3.5 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths,
Belgium
3.6 TIFF: Tagged Image File Format, a file of a gel image that can be analyzed in BioNumerics
3.7 PFGE Mailbox: An e-mail account that is maintained and checked by all database managers
at CDC. The address is: [email protected].
3.8 CDC Outbreak Response Team Mailbox: An e-mail account that is maintained and checked
by the outbreak response team at CDC. The address is: [email protected]
3.9 EDLB: Enteric Diseases Laboratory Branch
3.10 Area Laboratory: Laboratory, designated by CDC, which has agreed to assume responsibility
for additional PulseNet duties for laboratories within their support zone. The current Area
Laboratories include CDC, Massachusetts, Michigan, Minnesota, Texas, Utah, Virginia, and
Washington.
3.11 Cluster: A group of isolates, identified within the past 30 days for Yersinia; 60 days for
Salmonella, E. coli, Non-O157, Shigella, C. botulinum, Campylobacter, and Vibrio; and 120
days for Listeria, with the same serotype with indistinguishable PFGE DNA patterns by one
or more restriction enzymes
3.12 STEC: Shiga toxin-producing E. coli. STECs are the most virulent of the diarrheagenic
pathogens under surveillance in PulseNet.
3.13 NDA: A Non-disclosure Agreement is a legal contract between at least two parties that
outlines confidential materials or knowledge the parties wish to share with one another for
certain purposes, but wish to restrict from generalized use. In other words, it is a contract
through which the parties agree not to disclose information covered by the agreement. An
NDA creates a confidential relationship between the parties to protect any type of trade
secret. As such, an NDA can protect non-public business information.
VERSION: REPLACED BY AUTHORIZED BY:

Page 1 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

4. PROCEDURE:

4.1 SharePoint Access

4.1.1 All PulseNet personnel who wish to access SharePoint must send an email to the
PFGE Inbox ([email protected]) with “SharePoint Request” in the subject line.
4.1.1.1 The requestor should include the person’s first and last name, email address,
organization, phone number, job title and work address in the email. Complete
information is required to create an account so please include all requested
information.
4.1.1.2 The request email will be moved to the “Participants” folder by the database
manager who checks the PFGE Inbox.
4.1.2 Federal, state and local epidemiologists must request access to SharePoint through
the Outbreak Response Team at CDC [email protected] .
4.1.3 Access is approved if the person is certified and/or in the PulseNet contacts database.
If the person is not in the contacts database, contacts from the person’s lab will be
contacted by the designated database manager to assure the person should obtain
access.
4.1.4 If access is disapproved, the designated database manager notifies the requestor
immediately.
4.1.5 If access is approved, the PulseNet Database Unit Chief (or other personnel
designated by the Unit Chief) will add the participant to CDC Join. For additional
information on CDC Join, including how to add new participants please review the
training document saved here
\\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\SharePoint\CDC Join\CDC Join
Tips
4.1.6 Once a new participant has been added to CDC Join, they will receive a system-
generated email with instructions to complete registration and to send an email to the
sponsor, notifying them that registration has been completed. The PulseNet Database
Unit Chief (or other personnel designated by the Unit Chief) will submit an ITSO
ticket requesting the participant’s access to SharePoint. Make sure the ITSO ticket is
sent Attn: [PulseNet IT Support, see contacts section below for contact information]
and includes the new participant’s email address.
4.1.7 PulseNet IT support will be assigned the ITSO ticket and use the participant’s contact
information in CDC Join, http://externalpartners.cdc.gov, to populate a database. The
database will be used to keep track of the accounts, including NDA submission. In
case of an emergency and the SharePoint site is not accessible for an extended period
of time, the participants can be notified using the information in the database. Then
PulseNet IT support will grant access to SharePoint by adding their email to the
appropriate group. Once access to SharePoint has been granted, the participant will
receive a system-generated email welcoming them to SharePoint, providing login
information and requesting that they complete the Non-Disclosure Agreement
(NDA). When accessing the SharePoint site, for external partners, the username must
include the prefix cdcpartners\ and for CDC staff, the userID must include the prefix
cdc\.
4.1.8 Access to SharePoint will grant the new participant access to the Non-Disclosure
Agreement Subsite -. The new subscriber must print, read, sign, scan and email or fax
the completed NDA to CDC as described in Appendix PND03-1. Signing the NDA
VERSION: REPLACED BY AUTHORIZED BY:

Page 2 of 13
STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

indicates that the user agrees to follow the SharePoint policy on sharing information
(section 4.2 below).
4.1.9 When CDC receives the scanned or faxed copy of the completed NDA, EDLB
Administrative Support will add a check mark in the NDA section for the participant
and submit an ITSO ticket Attn: [PulseNet IT Support, see contacts section below for
name] stating the NDA has been received.
4.1.10 PulseNet IT Support will be assigned the ITSO ticket, verifies the account in the
database and closes the ITSO ticket.
4.1.11 Passwords must not be shared or disclosed to anyone, for security purposes.
4.1.12 When a participant leaves their current position or is no longer performing PulseNet
duties, they or their supervisor are required to notify CDC so that SharePoint access
is terminated. CDC may be notified by emailing [email protected] (or
[email protected] if epidemiologist) with “SharePoint” in the subject line
or by calling (404) 639-4558.
4.1.13 Once CDC is notified of a participant’s departure, the PulseNet Database Unit Chief
(or other personnel designated by the Unit Chief) submits an ITSO ticket Attn:
[PulseNet IT Support, see contacts section below for contact information] to have that
participant removed from SharePoint.
4.1.14 PulseNet IT Support receives the ITSO ticket, removes the participant from the
subscriber database, removes their access to SharePoint and closes the ITSO ticket.
4.2 SharePoint Policy on Sharing Information:
Often the PulseNet/OutbreakNet SharePoint postings contain preliminary information on
presumptive disease clusters and ongoing outbreak investigations. Therefore, SharePoint
postings are not appropriate for sharing with persons outside public health and food
regulatory agencies. If you would like to share SharePoint messages with persons not
directly associated with public health or food regulatory agencies, we require that you
obtain prior approval from the person or agency that posted the information. We would
appreciate your strict compliance with this policy. Violation of this policy will result in
loss of access to the PulseNet/OutbreakNet SharePoint website.
4.3 PulseNet/OutbreakNet SharePoint Administrative Roles:
There are four types of SharePoint users, each with a different level of privileges and
security. In descending order, these are the SharePoint Site Owner, Moderator, Member
and Visitor.
4.3.1 SharePoint Site Owners have complete control over the entire SharePoint website.
This level of administration is managed by the division SharePoint site collection
administrator, with oversight of the CDC Information Technology Services Office.
This is not managed by anyone in the PulseNet or Outbreak Units at CDC.
4.3.2 SharePoint Site Moderators have full responsibility for maintaining assigned
discussion forums by responding to, updating, archiving, and removing information
as described in 5.2. Moderators can add, edit or delete postings, retrieve them from
the archives, and modify discussion settings. This level of administration is managed
by the SharePoint site Owners.
4.3.3 SharePoint Site Members can read, post, and respond to messages on the
SharePoint site. Members can delete messages that they post. Members do not
necessarily have access to all discussions or subsites on the PulseNet/OutbreakNet
SharePoint site. This level of administration is managed by the SharePoint site
Owners.

VERSION: REPLACED BY AUTHORIZED BY:

Page 3 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

4.3.4 SharePoint Site Visitors can read messages on the SharePoint site. This level of
administration is managed by the SharePoint site Owners.
4.4 SharePoint Setup
4.4.1 SharePoint is divided into several organized content sections: Documents,
Announcements, Discussions and Subsites. Topics should be posted within the
appropriate content section. Topics may be added or removed at the discretion of the
Moderators.
4.4.2 Clicking on a topic within the content section allows the member to see the items and
postings within that document library, announcements section, discussion or subsites.

5. RESPONSIBILITIES:

5.1 Posting Cluster or Outbreak-Related Topics and Responses on SharePoint

5.1.1 Cluster and outbreak information should be posted on SharePoint for quicker and
more efficient reporting.
5.1.1.1 Indications for when to post a cluster
5.1.1.1.1 When a Salmonella or Shigella cluster of three or more indistinguishable
patterns has an epidemiological connection; state the epidemiological
link in the posting
5.1.1.1.2 When a Salmonella or Shigella cluster of three or more isolates (same
serotype) with a pattern that shows an increase in your state or region
5.1.1.1.3 For other organisms, when a cluster of two or more indistinguishable
patterns has an epidemiological connection; state the epidemiological
link in the posting
5.1.1.1.4 For other organisms, when there is a cluster of two or more isolates
(same serotype) with a pattern that shows an increase in your state or
region
5.1.1.1.5 When there is a cluster of a common pattern, monitor that cluster to see
if there is a significant increase in your laboratory before posting. A
significant increase can be determined by graphing out monthly uploads
of the pattern/pattern combination over time. Look at the frequency over
the past month, 60 or 120 days (depending on the organism) and
determine if it is an increase for that same time period, during that time
of year over the past several years.
5.1.1.1.6 Pattern and serotype commonality differ within each community,
therefore the decision to post a cluster is up to each individual laboratory
5.1.1.1.7 If you have questions about when to post, you can always contact CDC
or your PulseNet Area Laboratory
5.1.1.2 Isolate patterns must be uploaded to the PulseNet national databases before
posting to SharePoint (if not certified or unable to upload for any reason, the
patterns must be submitted via email to the PFGE inbox as soon as possible).
5.1.1.3 The following information must be included in the SharePoint posting and/or
sent by email to the PFGE Inbox (see Appendices PND03-2 and PND03-3 for
example postings):
5.1.1.3.1 For initial postings, a PulseNet bundle file containing the cluster or
outbreak patterns should be provided (TIFFs are no longer necessary)
5.1.1.3.1.1 A PulseNet bundle file will allow users to compare the isolates
in BioNumerics without having to reanalyze the gel image
VERSION: REPLACED BY AUTHORIZED BY:

Page 4 of 13
STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

and/or having to connect to the national database(s). NOTE: If a

PulseNet bundle file is not used, it will be deleted from
SharePoint by the discussion moderator and the poster will be
asked to post a PulseNet bundle file.
5.1.1.3.2 A subject line for the posting
5.1.1.3.3 Name of the submitting laboratory
5.1.1.3.4 Which enzyme(s) used—state if second enzyme is pending
5.1.1.3.4.1 Second enzyme should always be run for E. coli O157:H7 and
other STECs and Listeria monocytogenes per the “Second
enzyme recommendations” document posted on SharePoint.
5.1.1.3.4.2 Second enzyme should also be run on Campylobacter isolates
involved in a cluster or outbreak.
5.1.1.3.5 Serotype, if applicable
5.1.1.3.6 Shiga toxin type for E. coli O157 and other STECs if available
5.1.1.3.7 Collection and/or isolation date and date received when available
5.1.1.3.8 Information on additional isolates that may be part of the cluster or
outbreak
5.1.1.3.9 Additional information about the cluster pattern, such as number of
isolates and frequency of pattern in your local database
5.1.1.3.10 In an original posting, CDC pattern designations should not be posted by
anyone other than CDC Database Team members, even if the pattern is
listed as confirmed within the national database.
5.1.1.3.11 Postings should only include information pertaining to the laboratory’s
local data; regional data may only be included if a representative from
the included labs have been contacted first (additional observances or
questions regarding the national database should be limited to emails
directly to the PulseNet Database Team, [email protected])
5.1.1.3.12 Any available epidemiological information
5.1.1.3.13 Personal identifier information such as patient names should not be
posted to SharePoint
5.1.1.3.14 Please leave the Cluster Status field blank, the CDC Database Team will
complete this field. It will either be set to Active or Active (PN). Active
is defined as those clusters which are actively being followed by CDC
epidemiologists; Active (PN) is defined as those clusters which are not
actively being followed by CDC epidemiologists, but are considered by
the CDC PulseNet Database Team as worth watching.
5.1.1.3.15 Select an Interest group from the options in the pull down menu (Both,
Epi or Lab). This allows users to sort postings based on interest. For
example if the posting only contains laboratory data then select “Lab”; if
the posting contains information about a potential epidemiological link
between cases and/or an update on an epidemiological investigation then
select “Epi.” If the posting contains information related to both lab and
epi then select “Both.”
5.1.1.4 For laboratory response postings, the following information must be provided:
5.1.1.4.1 If a laboratory has seen the cluster pattern within the past 30 days for Y.
pestis, 60 days for E. coli, Non-O157, Shigella, Salmonella, C.
botulinum, Campylobacter, and Vibrio or 120 days for Listeria, then an
isolate number or file name should be provided in the posting.
VERSION: REPLACED BY AUTHORIZED BY:

Page 5 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

5.1.1.4.2 It is not necessary for a lab to post indistinguishable isolates that are
already included in a CDC cluster/outbreak summary spreadsheet on
SharePoint. Please continue to post new isolates that have not been
included in these spreadsheets.
5.1.1.4.3 It is mandatory that all patterns reported as indistinguishable be uploaded
to the PulseNet national databases or emailed to [email protected].
5.1.1.4.4 It should be noted that public health epidemiologists should go through
their laboratorians when contacting the CDC laboratorians regarding an
investigation.
5.1.1.4.5 Select an interest group from the options in the pull down menu (Both,
Epi or Lab). This allows users to sort postings based on interest. See
section 5.1.1.3.16 above for additional information.
5.1.1.5 Epidemiologist response postings
5.1.1.5.1 Epidemiologists are encouraged to post epidemiological information on
SharePoint. We encourage laboratorians and epidemiologists to work
together before posting information on SharePoint.
5.1.1.5.2 It should be noted that public health laboratorians should go through
their epidemiologists when contacting the CDC epidemiologists
regarding an investigation.
5.1.1.5.3 Select an interest group from the options in the pull down menu (Both,
Epi or Lab). This allows users to sort postings based on interest.
5.2 SharePoint Management
5.2.1 Thread Removal
5.2.1.1 Once a new thread has been posted to SharePoint, a discussion moderator
should respond within two business days of the original posting.
5.2.1.2 Moderator response postings for outbreaks or clusters should include (but are
not limited to) an outbreak/cluster code (if applicable), pattern number
assignments, frequencies of patterns, any pertinent epidemiological data
known, a PulseNet bundle file containing a pattern representative (if one has
not already been posted) and a line list.
5.2.1.3 Topics should be renamed with the following format: <Outbreak Code>
<(pattern number)>_<LabID of posting lab>_<Organism and/or Serotype>.
5.2.1.4 Only the information posted by the database team should be in dark purple
font.
5.2.1.5 Updated information should be posted by the discussion moderator as
necessary.
5.2.1.6 The discussion moderators reserve the right to delete all or part of any posting
if the criterion in section 5.1 is not met.
5.2.1.7 After 3 weeks of inactivity, if a cluster code was assigned, a cut thread will be
posted to the topic indicating that the posting will be removed after a week
unless someone responds with a reason why the posting should not be archived
(i.e. epi information, local or regional increase, etc.). The original posting and
all response postings will be archived according to the “PulseNet Guidelines
for Archiving SharePoint Postings” (see Appendix PND03-4).

6. FLOW CHART:

7. BIBLIOGRAPHY:
VERSION: REPLACED BY AUTHORIZED BY:

Page 6 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

8. CONTACTS:

8.1 PulseNet Database Team

[email protected]
(404) 639-4558
8.2 OutbreakNet Response Team
[email protected]
8.3 PulseNet Database Unit Chief
Kelley Hise
[email protected]
(404) 639-0704
8.4 EDLB Administrative Support
Mike Korth
[email protected]
(404) 639-2099
8.5 PulseNet IT Support
Brenda Brown
[email protected]
(404) 639-3942

9. AMENDMENTS:

9.1 This SOP replaces the former SOP PND03 PulseNet Listserv Access and Use describing
the use of WebBoard as the PulseNet communication listserv. As of February 29, 2008 the
contents of WebBoard were moved to the PulseNet workspace on CDC Team. CDC Team
is now being used as the PulseNet web-based discussion forum.
9.2 2011-02-22 Appendix PND03-4 was updated
9.3 2011-07-27 Appendix PND03-4 was updated
9.4 2011-08-29 5.2.1.5 and 5.2.1.6 were updated to clarify when postings would be removed
from CDC Team
9.5 2011-09-09 Section 5.2.1 and appendix PND03-4 were updated. Previously database
managers would post a cut thread to all postings with at least 3 weeks of inactivity and
then archive the posting once the timeframe in the cut thread had passed. Please see section
5.2.1 and appendix PND03-4 for updated guidelines for archiving postings.
9.6 This SOP replaces the former SOP PND03 PulseNet Listserv Access and Use describing
the use of CDC Team as the PulseNet communication listserv. As of December 19, 2011
the contents of the PulseNet workspace on CDC Team were moved to the
PulseNet/OutbreakNet SharePoint site. SharePoint is now being used as the PulseNet web-
based discussion forum.

VERSION: REPLACED BY AUTHORIZED BY:

Page 7 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

Appendix PND03-1

PulseNet/OutbreakNet SharePoint Site Non-disclosure Agreement

Welcome to the PulseNet/OutbreakNet SharePoint site!

In order to comply with Health and Human Services security requirements, we are required to obtain a
signed non-disclosure agreement (NDA) from each PulseNet/OutbreakNet SharePoint site participant.

The following is the PulseNet SharePoint and OutbreakNet site policy on sharing information. It is
displayed on the PulseNet/OutbreakNet SharePoint site as a reminder:

PulseNet SharePoint and OutbreakNet site Policy on Sharing Information

Often PulseNet/OutbreakNet SharePoint postings contain preliminary information on presumptive

disease clusters and ongoing outbreak investigations. Therefore, SharePoint postings are not
appropriate for sharing with persons outside public health and food regulatory agencies. If you
would like to share SharePoint messages with persons not directly associated with public health
or food regulatory agencies, we require that you obtain prior approval from the person or agency
that posted the information.

We would appreciate your strict compliance with this policy. Violation of this policy will result in
loss of access to the PulseNet/OutbreakNet SharePoint site.

Thank you for your cooperation

Peter Gerner-Smidt MD, PhD

PulseNet, Centers for Disease Control and Prevention

Ian Williams, PhD, MS

OutbreakNet, Centers for Disease Control and Prevention

VERSION: REPLACED BY AUTHORIZED BY:

Page 8 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

***** ***** ***** E-MAIL THIS PAGE ONLY ***** ***** *****

PulseNet/OutbreakNet SharePoint Non-Disclosure Agreement

By signing this non-disclosure agreement, you agree that you will not share information from the
PulseNet/OutbreakNet SharePoint site with personnel not associated with the public health community
without appropriate approval.

I have read the PulseNet/OutbreakNet SharePoint Policy on Sharing Information and agree not
to share information from the PulseNet/OutbreakNet SharePoint site with personnel not associated with
the public health community without appropriate approval.

Name:

PulseNet/OutbreakNet Affiliation:

Email Address:

Signature: Date:

Sign, Scan and email to: Mike Korth at [email protected]

VERSION: REPLACED BY AUTHORIZED BY:

Page 9 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

Appendix PND03-2

Example: Correct PulseNet Initial SharePoint Posting

Discussion: E. coli

Subject: E. coli O157:H7 in GA

Body: GA is experiencing a cluster (5 cases and 1 food) of E. coli O157:H7 in the Atlanta area. The
following information pertains to the attached PulseNet bundle file, GA110374PN. The associated TIFF
was uploaded to the PulseNet national database for E. coli this morning. Both enzymes have been run for
these isolates.

Collection Source Type Source Site

Isolate # Date received
date
GA1000 01-05-11 01-08-11 Human Stool
GA1001 01-04-11 01-07-11 Human Stool
GA1002 01-08-11 01-11-11 Human Blood
GA1003 01-08-11 01-10-11 Food Hamburger
GA1004 01-06-11 01-08-11 Human Stool
GA1005 01-03-11 01-06-11 Human Blood

[Patient demographics, including age, sex, and county, is not necessary in SharePoint posting, but needs
to be included in the email to [email protected] if information has not been uploaded to the PulseNet
database.]

Within our local database, this pattern is not common; we have seen it only once before. The isolate
number is GA0076, and it was uploaded in January of 2008. As of this date, the cluster is potentially
linked to hamburger.

Epi Contact Info: John Doe [email protected]

***GA11374PN.BDL*** (this is the attached PulseNet bundle file)

Cluster Status: Please leave this blank, the CDC database team will add this information.

Interest Group: Both (indicates both lab and epi may be interested in this posting)

VERSION: REPLACED BY AUTHORIZED BY:

Page 10 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

Appendix PND03-3

Example: Correct PulseNet Response SharePoint Posting

If lab has seen posted pattern:

Discussion: E. coli

Subject: E. coli O157:H7 in GA

Body:
AL has two possible matches to the GA patterns. These isolates are AL1234 with an upload date of 1-12-
11 and AL1245 with an upload date of 1-13-11.

***AL11056PN.bdl*** (this is the attached PulseNet bundle file)

Interest Group: Lab (indicates lab will be interested in this posting)

VERSION: REPLACED BY AUTHORIZED BY:

Page 11 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

Appendix PND03-4

PulseNet Guidelines for Archiving SharePoint Postings

1. If a SharePoint cluster posting does not follow the posting guidelines as stated in section 5.1 of the
SOP, the database manager reserves the right to edit or delete the posting. The poster should be
notified that the posting is being deleted and why. The poster should also be reminded of when and
when not to post a cluster to SharePoint.

2. Depending on the pathogen/database, SharePoint postings that receive cluster codes are archived 4
weeks from the LAST posting of the topic or thread and/or if the epis decide to close the cluster.

3. Postings that do not get a code, may be kept on SharePoint for a longer period of time if the national
and/or regional numbers are indicating a potential rise or due to other reasons as determined by the
national database managers.

4. A cut thread should be posted for all clusters. If the cluster has been closed for a while, either by the
epis or on SharePoint, no cut thread needs to be posted.

5. Only for larger outbreaks, and especially those that resulted in regulatory action--before posting the
cut thread, summarize the information from the posting. (i.e., if it was an outbreak, give pattern
numbers, numbers seen from which state(s), commonality of the pattern in the database, if the
outbreak was ever linked back to a food item and/or resulted in a recall). Or you could simply post the
link to the CDC web update if there is one. A template can be found here:
\\cdc\project\CCID_NCZVED_DFBMD_PulseNet\SharePoint\admin\Templates

6. Post a cut thread (see below or go to

\\cdc\project\CCID_NCZVED_DFBMD_PulseNet\SharePoint\admin) to that posting.

[title topic] has been closed and will be removed from SharePoint in approximately 7 days. Any
additional replies or inquiries should be made before then.

Thank you,

PulseNet Database Administration Team

Phone: (404) 639-4558

7. NOTE: the above can be done automatically from the outbreak log:
A) Before you close a cluster, click on the “Close Clusters” button in the main organism screen
B) Choose the cluster(s) you want to close and move them to the Clusters to Close box
C) Click the button “Close and Generate Cut Thread(s).”

8. After 7 days, if there have been no responses, the topic can be archived.

9. To archive a posted thread:

• Highlight the entire topic—make sure to include all postings—and copy
VERSION: REPLACED BY AUTHORIZED BY:

Page 12 of 13
 STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE CODE: PND03
Effective Date:
PULSENET AND OUTBREAKNET SHAREPOINT SITE 08 15 14

• Go to Edit>Paste Special, and choose “Unformatted Text”, and paste the copied topic into a blank
Word document
• Save the word document under:
\\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\SharePoint\Forum\topic folder\year
• Use the following format for the filename: for coded outbreaks and clusters that do not get codes
use: Topic name (ex: 0707COEXK-1_(EXKX01.0001)_CO_E. coli O121/O26) or for non-outbreaks
use: YYYY-MM-DD_LabID-Topic (ex: 2002-09-17_CDC-Serotyping and Ribotyping
Outbreak.doc); the date is the date of the first posting
• Save only attachments that we would not already have elsewhere (TIFFs and bundles in the database;
line lists in the outbreak folder, etc.) into an appropriate “attachments” folder.

10. Once the thread is saved, the thread can be deleted from SharePoint.

VERSION: REPLACED BY AUTHORIZED BY:

Page 13 of 13
 CODE: PND04
STANDARD OPERATING PROCEDURE FOR PFGE GEL ANALYSIS Effective Date:
07 22 2011

1. PURPOSE: To describe the guidelines for standardization of TIFF image analysis in order to accurately
compare PFGE patterns between PulseNet participating laboratories.

2. SCOPE: This procedure applies to all analysis submitted to PulseNet, thereby allowing comparisons of
results with other PulseNet laboratories.

3. DEFINITIONS/TERMS:

3.1 PFGE: Pulsed-Field Gel Electrophoresis

3.2 TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
3.3 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
3.4 SOP: Standard Operating Procedure

4. BACKGROUND: PulseNet Gel Analysis Guidelines

Computerized gel analysis with BioNumerics facilitates comparison of PFGE patterns from isolates of foodborne
bacteria on gels produced by all certified PulseNet laboratories. It is critical that excellent quality gels that have been
analyzed accurately are submitted to the PulseNet on-line databases for valid comparison of results.

Computer analyses (from lane definition to normalization to band finding) should always be checked against a copy
of the actual gel image being analyzed, as visual (i.e., manual) analysis is the gold standard. Analysis is only as good
as the TIFF of the gel image being analyzed. For this reason, each gel image should be of the highest quality
possible. Consistency from run to run is critical. Factors such as fuzzy bands, smearing, high background, the
presence of shadow bands, and running the gel too short or too long can make TIFFs of the images needlessly
difficult to analyze and can compromise comparisons to patterns in the national databases. Gels that do not yield
TIFFs with acceptable quality should be rerun.

Sometimes, suggestions to improve gel quality come about after computer analysis of what appears to be a good gel
visually. Factors such as consistent slants of lanes and bands, band distortions, light bands at the bottom of the gel or
in a few lanes, extreme differences in the band spacing of the standards compared to the global standard, debris or
artifacts that affect analysis, and poor band resolution can be more apparent during computer analysis of a TIFF than
upon visual inspection. When factors such as these are noted during analysis, the person performing the analysis
should suggest and/or implement changes in gel preparation so that gel quality will be of the highest quality
possible.

5. RESPONSIBILITIES/PROCEDURE:

5.1 Standards
5.1.1 Standards are very important for normalization. Standards can also be used to gauge consistency from
gel to gel and provide clues about how to evaluate the test isolates. Evaluation of the standards may
include the following:

5.1.1.1 Are the standard bands consistently the same thickness or intensity from gel to gel?
5.1.1.2 During normalization, note how the standard pattern on the gel matches the band pattern of the
reference standard. If BioNumerics must stretch or shrink the pattern to match the reference
standard, is the stretching or shrinking the same from gel to gel? If a sudden change occurs, an
error might have occurred during the run such as the wrong electrophoresis program used to run
the gel. Using the show distortion bars feature will aid in visualizing these types of problems and
assist with correct normalization.
5.1.1.3 From gel to gel, is the standard pattern consistently the same or does the gel resolution vary
enough to affect the standard pattern (i.e., doublets appear sometimes and single bands appear at
other times in certain areas of the pattern)? For normalization purposes on E. coli O157:H7 gels
(for example), 15 normalization markers on 15 single bands can be placed on the Salmonella
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 5
 CODE: PND04
STANDARD OPERATING PROCEDURE FOR PFGE GEL ANALYSIS Effective Date:
07 22 2011

Braenderup H9812 standard. However, some laboratories are resolving doublets where single
bands appear on the global standard near the bottom of the H9812 pattern. If doublets resolve,
mark the normalization bands carefully.
5.1.2 Not all observed bands in a standard lane are used to normalize. If an area of a standard lane resolves as a
doublet on a test lane but appears as a single band on the global standard, a single normalization marker
should be marked in this area on the test lane. However, for band finding (step 4), a doublet should be
marked on the test lane. Normalization (step 3) will not be affected if areas of the standard lanes are
normalized correctly as single bands but marked as doublets during band finding.
5.1.3 If shadow bands appear in the standard lanes, shadow bands could be present in test lanes. Ideally, the gel
should be repeated. Lanes containing shadow bands are difficult to analyze. Shadow bands probably
indicate incomplete restriction and should not be present on a gel. Repeat the gel performing restriction
with more units of enzyme, for a longer amount of time and/or with a different lot of enzyme. Wash the
plugs at least two more times with TE Buffer before restriction is repeated.

5.2 Before analysis

5.2.1 Consistently taking pictures where the gel image fills almost the entire window on the imaging equipment
screen (without cutting off wells or the bottom of the gel) should improve consistency in normalization
and band finding. Please see the SOP PNL07 Image Acquisition for instructions and additional
information.
5.2.2 Print out or open up on a computer screen the gel image to be analyzed. A printed copy of the gel image
can help steer decisions on band finding, such as deciding if an area is a doublet or a single band.
5.2.3 If shadow bands are present or if incomplete restriction is suspected, rerun the sample(s) or, if necessary,
the entire gel. Lanes with incomplete restriction of DNA are difficult to analyze and may yield a different
pattern when the PFGE is repeated. Results from isolate patterns showing incomplete restriction are not
always reproducible and cannot be accurately compared with patterns from other isolates in a local
database or with the national database.

5.3 BioNumerics – Step 1 Strips

5.3.1 At the beginning of the analysis, assign the TIFF to the enzyme used on the gel. If two enzymes are used,
lanes restricted with secondary enzymes should be changed after analysis but before lane linkage to the
local database (see section 5. 7of this document).
5.3.2 To define the gel strips, place the sides of the green box frame on the edges of the first and last lanes of
the gel. Place the top of the green box frame directly under the wells. Place the bottom of the green box
frame approximately 1-2 cm below the lowest band of the Salmonella Braenderup H9812 standard on a
full-size image. (On a gel following the PulseNet standardized procedure where the last band of the
standard is 1-1.5 cm from the bottom of the gel and the gel image fills the entire window on the imaging
equipment screen, 1-2 cm below the last band of the standard should be at the bottom of the gel.) This
will standardize lane lengths, which will produce more accurate normalizations and improve comparisons
with the national database.
5.3.3 After the lanes are defined, adjust the “thickness” under “edit settings” to increase or decrease the
thickness of the defined gel strips so that the left and right edges of the strips are just outside the outer
edge of the bands. This will ensure that the bands will appear in a size for proper analysis. If the gel strips
are defined incorrectly (too wide), the resulting narrow bands with large white patches on either side of
the bands are difficult to analyze. Gel strips may need adjustment when switching between 10-well and
15-well gels and also if the gel image is captured differently (i.e., close up versus farther away) from gel
to gel. The width of an individual lane can be adjusted independently, if necessary.
5.3.4 Using the “linear adjustment” in the “edit tone curve” feature may provide better band clarity for
analysis.
5.3.5 In the edit tone curve feature, enhancing weak bands is sometimes useful to detect bands accurately.
However, when using the edit tone curve feature to enhance weak bands (primarily seen at the bottom of
the gel), too much enhancement of weak bands may make the stronger stained bands in the top and
VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 5
 CODE: PND04
STANDARD OPERATING PROCEDURE FOR PFGE GEL ANALYSIS Effective Date:
07 22 2011

middle portions of the pattern appear too thick and make them difficult to analyze. Always compare band
markings in the software to a copy of the original gel image.
5.3.6 Increasing contrast is sometimes useful to detect bands accurately.
5.3.7 Do not use background reduction or spot removal on gel images submitted to CDC. The different usage
of these BioNumerics tools among PulseNet participating laboratories causes problems when performing
comparisons within the national databases. Participating laboratories may use background reduction and
spot removal in their local databases, but should remove these features before submitting analyses to the
national databases. CDC does not recommend the use of spot removal.

5.4 BioNumerics - Step 2 Curves

5.4.1 If bands in the test lanes or the standard lanes are distorted or contain artifacts and the gel image must be
analyzed without rerunning the gel, ensure that the curve strips are placed accurately.

5.5 BioNumerics - Step 3 Normalization

5.5.1 If artifacts (e.g., specks or dots) appear near a band on the TIFF image, ensure a normalization marker is
not placed on the artifact instead of where the actual band is on the TIFF image.
5.5.2 Check normalization using the “distortion bars” function (under the Normalization menu). The color of
the distortion bars should be the same moving across the gel. If a high amount of orange or red distortion
bars or distortion bar colors that change as they move horizontally across the gel strips appear,
normalization marker placement should be examined. If dark distortion bars are present and uneven
across the gel and the normalization marker placement is correct, the gel should be rerun, verifying that
the correct running conditions are used.

5.6 BioNumerics - Step 4 Bands

5.6.1 If you use the auto search function, look at the whole gel and delete obvious extra bands first before
carefully going through each lane and matching band markings to a copy of the gel image. This will
ensure that extra bands, such as those near the top or bottom of a TIFF image, are deleted. If artifacts are
present on a TIFF image or the TIFF image is dark, the auto search function may find many extra bands.
5.6.2 Review the bands detected by the auto search function.
5.6.2.1 Toggle between the normalized and un-normalized viewing screens to ensure bands were not
accidentally marked above and/or below the standard. Sometimes debris will be marked
outside of the normalized viewing screen. This is important to double check.
5.6.2.2 Mark bands carefully. Use the peak of the shoulder on the densitometric curve if band
placement is in question. If a band is considered a singlet, ensure that the single band is placed
correctly. Bands should be marked in the middle of areas considered single, solid bands.
5.6.2.3 Mark bands as they appear on a copy of the actual gel image. Defer to a hard copy of the image
when determining if a questionable area should be marked as a doublet or a single band.
5.6.2.4 All lanes containing profiles, which by visual inspection are obviously indistinguishable,
should be marked uniformly.
5.6.2.5 If artifacts (e.g., specks or dots) appear near a band on the TIFF image, make sure a band is not
placed on the artifact instead of where the actual band is on the gel image.
5.6.3 When deciding if a band is a doublet or a single band, look for white space in the middle of the area or
indentations (i.e., doughnuts or shoulders) on the sides of the area that separate two bands. If indentations
or white space are apparent on a copy of the TIFF, the area is most likely a doublet. If the indentations or
white space are questionable, the area probably should be marked as a single band. Separate peaks on the
densitometric curve may also provide clues as to the band designation of the area.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 5
 CODE: PND04
STANDARD OPERATING PROCEDURE FOR PFGE GEL ANALYSIS Effective Date:
07 22 2011

Single band Doublet

5.6.4 The uncertain band feature of BioNumerics should be used sparingly. If an area on an excellent or good
gel is either a doublet or a single band and the criteria in 5.6.3 does not aid in band marking, the
uncertain band feature may be used. If the gel quality is fair or poor, repeat the gel and produce a good
quality pattern before marking bands.
5.6.5 As overall gel quality improves, resolution will become sharper. If an area that previously resolved as a
thick singlet becomes a doublet or triplet on subsequent gels, that area should be marked exactly as it
appears on each gel.
5.6.6 Visible bands of test isolates should be marked down to ~20.5 kb, the bottom band used for
normalization in the Salmonella Braenderup H9812 standard. If any portion of a band is even with or
above the bottom of H9812 it should be marked. If the entire band falls below the last band of the
standard, it should not be marked. There will always be some level of variability with PFGE, use your
best judgment and please remember to be consistent.

Should be marked Should not be marked

5.6.7 In almost all cases, band intensity differences are not reliable enough for use in assigning pattern
numbers. Two patterns that are indistinguishable except for one or more band intensity difference(s) will
be assigned the same pattern number. Isolates with only intensity differences should be reported as
indistinguishable by the test(s) run and the PulseNet criteria.

5.7 Lane linkage

5.7.1 Before linking a lane to the local database, make sure that the fingerprint type (e.g., PFGE-XbaI, PFGE-
BlnI, etc.) is correct. Changing fingerprints is necessary when more than one enzyme is used for
restriction on a particular gel. If the lanes are linked with incorrect fingerprint types, possible duplicate
entries in the database could occur. At the beginning of the analysis, assign the TIFF to they enzyme used
on the gel. Any lanes restricted with secondary should be changed after analysis but before lane linkage
to the local database. To change the fingerprint type, right-click on the desired lane and select “Change
fingerprint type of lane…” After the fingerprint type is changed, you can link the lane to an entry in the
database. Both fingerprint types should be indicated with a green dot next to the one entry in the
BioNumerics database.

5.8 Review of analysis and submission to CDC

5.8.1 PulseNet participating laboratories should review their band finding and normalization before submitting
to the PulseNet national database, PFGE inbox, or the PulseNet Listserv. After band finding and the
linkage of lanes in a TIFF, normalization and band finding can be roughly checked by analyzing a
dendrogram showing gel strips and band markings.
5.8.2 Submit your laboratory’s best possible gel image and analysis of a particular pattern. If one or more lanes
of an otherwise good gel are unsatisfactory, do not upload the unsatisfactory lanes to the national
database. Rerun unsatisfactory isolates on another gel.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 5
 CODE: PND04
STANDARD OPERATING PROCEDURE FOR PFGE GEL ANALYSIS Effective Date:
07 22 2011

5.8.4 Within your laboratory, regularly check band markings of all analysis-certified personnel to ensure
consistency. This should be included as part of your laboratory’s Quality Assurance/Quality Control
program.
5.8.5 All TIFF and bundle files must be named according to the PulseNet protocols for naming. TIFFs and
bundle files from each PulseNet participating laboratory should have unique names by laboratory and not
by species when they are sent to CDC. For example, the first TIFF and bundle from GA in 2011 should
be named GA11001.tif and GA11001PN.bdl. This could be a gel containing E. coli O157:H7 or another
organism. The second TIFF and bundle from GA in 2011 should be named GA11002.tif and
GA11002PN.bdl. This could be a gel containing Salmonella or another organism.
5.8.6 All bundle files posted to the PulseNet Listserve and/or exchanged with PulseNet participating
laboratories must be created using the Create PulseNet Bundle file lightning bolt icon on the left side of
the BioNumerics screen. Bundle files created using this icon will contain only PulseNet fields and “PN”
will automatically be added to the file name. Do not ever manually add “PN” to the end of a non
PulseNet bundle file.

5.9 After submission to CDC

5.9.1.1 All certified PulseNet participating laboratories have the ability to check the on-line databases
to see if previously submitted patterns have been assigned pattern numbers by CDC. These
pattern names can be downloaded to the local database.
5.9.1.1.1 If a pattern number is followed by a “&” or “@,” the number has not been confirmed by
the CDC Database Team. These pattern numbers are to be considered preliminary and
must not be reported.
5.9.1.1.2 If the band marking is different in the local database than it is in the on-line database,
the CDC Database Team has changed the participating laboratory’s original analysis.
The participating laboratory should review the changes made by the Database Team,
and if necessary, contact CDC to find out why the changes were made. The participating
laboratory can then download the changes to their local database.
5.9.1.1.3 If a pattern status has been marked “unsatisfactory,” the submitting laboratory must
rerun the isolate on another gel. Contact the CDC Database Team if there is a question
about the designation.
5.9.2 If resubmission/reupload of results from the same TIFF is necessary (e.g., for Salmonella serotype
information), highlight all isolates on that gel before reuploading, even if only one lane has results that
are being resubmitted. If all lanes are not highlighted, unlinking could occur.

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:

9. AMENDMENTS:

9.1. Statements regarding unsatisfactory patterns having “NG” in the pattern names were removed because the
CDC Database Team no longer uses ‘NG” to denote an unsatisfactory pattern. Please see section 5.9.1.2.3.
9.2. Updated section 5.8.5 and 5.8.6 to include a description of the “PN” for PulseNet bundle files
9.3. References to the BioNumerics Training Manual were removed. Current versions of the training
notebooks are posted on the PulseNet Listserv after each CDC BioNumerics Training Course. Please refer
to those presentations for step-by-step instructions on using the tools in BioNumerics.
9.4. 2011-07 Section 5.6.6 was updated to clarify verbiage and add example images.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 5
 STANDARD OPERATING PROCEDURE FOR SETUP AND USE OF SECURID KEY CODE: PND08
Effective Date:
FOB 5 09 2005

1. PURPOSE: To describe the procedure for setup and use of a SecurID Key Fob.

2. SCOPE: This procedure applies to all PulseNet personnel who are analysis certified and receive a
SecurID Key Fob.

3. DEFINITIONS/TERMS:

3.1. SecurID Key Fob: Token that displays a six-digit passcode. When used in combination with a
four-digit pin number, allows access through the CDC firewall.

4. RESPONSIBILITIES/PROCEDURE:

4.1 Precautions for Protecting Your SecurID Key Fob. For your own protection and that of the
system, always take the following precautions:
4.1.1 Never reveal your PIN or user password to anyone. Do not write them down.
4.1.2 If you think someone has learned your PIN, notify CDC PulseNet to have the PIN cleared
immediately. At your next login you will have to create a new PIN to use.
4.1.3 Exercise care not to lose your SecurID Key Fob or to allow it to be stolen. If your key fob
is missing, notify CDC PulseNet immediately. They will contact the administrator, who
will disable the key fob so that it is useless to unauthorized users.
4.1.4 Do not let anyone access the system under your identity (that is, log in with your PIN and a
code from your SecurID token).
4.1.5 It is essential to site security that you follow your system’s standard logoff procedures.
Failure to log off properly can allow unauthorized access from your workstation by another
user.
4.1.6 Protect your key fob from physical abuse. Do not immerse it in liquids or expose it to
extreme temperatures.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 8
 STANDARD OPERATING PROCEDURE FOR SETUP AND USE OF SECURID KEY CODE: PND08
Effective Date:
FOB 5 09 2005

Setting Up a New PIN

1. Open a web browser, either Netscape or Explorer.

2. Access the CDC SecurID System website by entering the following address for location:
http://securid.cdc.gov

3. Select Set new PIN.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 8
 STANDARD OPERATING PROCEDURE FOR SETUP AND USE OF SECURID KEY CODE: PND08
Effective Date:
FOB 5 09 2005

Assigned by CDC

4. Enter your Username:

You have received your username in a separate communication.

NOTE: Please be sure there are five or six “ticks” on the left side of the device. This will allow time for
synchronization between the device and the server for authentication.

5. Click Send.

If you receive this screen again, please wait for the next number on the SecurID Key Fob, then enter the
username and number.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 8
 STANDARD OPERATING PROCEDURE FOR SETUP AND USE OF SECURID KEY CODE: PND08
Effective Date:
FOB 5 09 2005

6. Enter the PASSCODE: Initially, this is the six digits displayed on the SecurID Key Fob.

7. Click Send.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 8
 STANDARD OPERATING PROCEDURE FOR SETUP AND USE OF SECURID KEY CODE: PND08
Effective Date:
FOB 5 09 2005

8. Enter a four digit number that will serve as your PIN.

9. Then enter your PIN again for verification.

10. Click Send.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 8
 STANDARD OPERATING PROCEDURE FOR SETUP AND USE OF SECURID KEY CODE: PND08
Effective Date:
FOB 5 09 2005

11. Your PIN has been accepted. Wait for the numbers on the SecurID Key Fob to change, then
enter your PASSCODE. (Now it is your four digit PIN + six digits displaying on SecurID Key
Fob – no spaces.)

12. Click Send.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 8
 STANDARD OPERATING PROCEDURE FOR SETUP AND USE OF SECURID KEY CODE: PND08
Effective Date:
FOB 5 09 2005

You have now set up and tested your PIN.

NOTE: Please be sure to read and observe Precautions for Protecting Your SecurID Key Fob and the
secured information you will be accessing.

NOTE: If you leave your current position, notify CDC PulseNet and return the SecurID Key Fob to
FedEx to the following address:
Attn: Michael Korth
Centers for Disease Control
1600 Clifton Road, NE
MS C03
Atlanta, GA 30333
Phone: (404) 639-3334

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 8
 STANDARD OPERATING PROCEDURE FOR SETUP AND USE OF SECURID KEY CODE: PND08
Effective Date:
FOB 5 09 2005

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:
7.1 PulseNet Database Team
Centers for Disease Control and Prevention
1600 Clifton Road NE
MS C03
Atlanta, Georgia 30333
Phone: (404) 639-4558
Email: [email protected]

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 8
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

1. PURPOSE: To describe the procedure for authenticating and connecting to the server.

2. SCOPE: This procedure applies to all PulseNet personnel who are analysis certified and receive a
SecurID Key Fob.

3. DEFINITIONS/TERMS:

3.1. SecureID Key Fob: Token that displays a six-digit passcode. When used in combination with a
four digit PIN number, allows access through the CDC firewall.
3.2. BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium.

4. RESPONSIBILITIES/PROCEDURE:

Step 1: Authenticate to the Firewall

Open a web browser, either Netscape or Internet Explorer

Access your bookmark name Authentication Form or enter the following address for location:
http://gateway-sdn.cdc.gov:900.
You will see this screen: Client Authentication Remote Service

VERSION: REPLACED BY AUTHORIZED BY:

Page 1 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

Enter your login. You will receive your username in a separate communication.

Click Submit.

You will see this screen: Client Authentication Remote Service

Enter your 10-diigit password:

four-digit PIN plus six-digit SecurID number

NOTE: Please be sure there are two or three “ticks” on the left side of the device. This will allow time for
sychronization between the device and the server for authentication.

Click Authentication.

VERSION: REPLACED BY AUTHORIZED BY:

Page 2 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

You will see this screen: Client Authentication Remote Service

Verify Standard Sign-on is selected.

Click Submit.

VERSION: REPLACED BY AUTHORIZED BY:

Page 3 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

You will see this screen: Client Authentication Remote Service

You should have at least one rule.

Minimize the screen

NOTE: IF YOU CLOSE THIS SCREEN YOU WILL TERMINATE YOUR CONNECTION TO THE
FIREWALL.

VERSION: REPLACED BY AUTHORIZED BY:

Page 4 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

Step 2: Using BioNumerics

BioNumerics
v3.0

Open BioNumerics

VERSION: REPLACED BY AUTHORIZED BY:

Page 5 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

Select the database you want to analyze (this could be Ecoli-client, Salmonella-client, Listeria-client,
Shigella-client, or Campylobacter-client).

Click Analyze

VERSION: REPLACED BY AUTHORIZED BY:

Page 6 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

The PulseNet logo will appear if you have installed the PulseNet customization files.

This screen tells you the total number of isolates currently in your database, then the individual number,
by pattern.

VERSION: REPLACED BY AUTHORIZED BY:

Page 7 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

To connect to the Online BioNumerics server:

Click Database

Select Connect to server…

REMINDER: YOU SHOULD STILL HAVE THE AUTHENTICATION

SCREEN MINIMIZED.

VERSION: REPLACED BY AUTHORIZED BY:

Page 8 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

Enter the IP address of the BioNumerics server. You have received this in a separate communication.

Verify the Port number is 7013

Click OK

VERSION: REPLACED BY AUTHORIZED BY:

Page 9 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

Enter the Login and Password to access the BioNumerics server. You have received this in a separate
communication.

Click OK

VERSION: REPLACED BY AUTHORIZED BY:

Page 10 of 12
 STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

You will see the IP address and the server login

You have now successfully connected to the BioNumerics Online server.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

7.1 CDC PulseNet Database Team

Centers for Disease Control and Prevention
1600 Clifton Road NE
Mailstop C03
Atlanta, Georgia 30333
Phone: (404) 639-4558
Email: [email protected]

8. AMENDMENTS:

VERSION: REPLACED BY AUTHORIZED BY:

Page 11 of 12
STANDARD OPERATING PROCEDURE FOR AUTHENTICATING AND CODE: PND09
Effective Date:
CONNECTING TO THE SERVER 5 09 2005

VERSION: REPLACED BY AUTHORIZED BY:

Page 12 of 12
 STANDARD OPERATING PROCEDURE FOR BIONUMERICS SOFTWARE CODE: PND10
Effective Date:
TRAINING 5 09 2005

1. PURPOSE: To describe the procedure for training PulseNet personnel in PulseNet database
management and communication protocols.

2. SCOPE: This procedure applies to all PulseNet participants and hosts of PulseNet database training
courses.

3. DEFINITIONS/TERMS:

3.1 Host lab: Term used to describe a PulseNet laboratory that has been approved by CDC to host a
training course
3.2 Training personnel: Term used to describe PulseNet participant(s) who have been approved by
CDC to train other PulseNet participants
3.3 APHL: Association of Public Health Laboratories
3.4 SOP: Standard Operating Procedure
3.5 CDC: Centers for Disease Control and Prevention
3.6 TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
3.7 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium

4. RESPONSIBILITIES:

4.1 All PulseNet personnel are required to read the PulseNet QA/QC manual and all PulseNet SOPs.
4.2 At least one PulseNet participant from each participating PulseNet laboratory is required to
attend annual PulseNet update meetings and regional meetings when they occur.
4.3 All PulseNet personnel submitting TIFFs of PulseNet pathogens for submission to the national
databases must have at least a basic level of computer knowledge and be familiar with the
BioNumerics analysis software.
4.4 Host lab(s), APHL, and/or CDC will determine training needs of PulseNet participants.
4.5 All PulseNet personnel must be trained by a host lab or by other approved training personnel.
4.5.1 PulseNet database management and communication training covers analysis of PFGE
gels using BioNumerics software, how to access the PulseNet National Databases with
data-sharing tools, how to upload PFGE data to the national server, and how to
communicate with others in the PulseNet network and CDC.
4.6 Host labs and training personnel should adequately prepare participants in the PulseNet database
management and communication. When training is finished, the trainee(s) should be able to
submit PulseNet gels and bundle files for analysis certification (see SOP PNQ02).
4.7 Host labs and training personnel must be analysis-certified.
4.8 Host labs must put together training materials for the trainees.
4.9 PulseNet participants will then be evaluated through certification (PNQ02) and proficiency
testing (PNQ04).

5. PROCEDURE:

5.1 APHL and CDC continuously monitor PulseNet participant training needs.
5.2 Host labs and/or training personnel should work with trainees and/or APHL to determine a
feasible time and location for the course.
5.3 The following is a recommended procedure for hosting a PulseNet database management course:
5.3.1 The host lab should organize an agenda committee to create an agenda and a timeline of
organizational duties, obtain needed training materials, and arrange for IT support.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 3
 STANDARD OPERATING PROCEDURE FOR BIONUMERICS SOFTWARE CODE: PND10
Effective Date:
TRAINING 5 09 2005

5.3.2 It is recommended that each participant be provided, but not limited to, the following
items:
5.3.2.1 Participant and trainer contact information
5.3.2.2 Handouts of training presentations, preferably in an organized notebook format
5.3.2.3 CD containing the BioNumerics software (for use in class only)
5.3.2.4 CD containing PulseNet masterscripts (for use in class only)
5.3.2.5 Practice exercises and training TIFFs with all PulseNet organisms
5.3.2.6 Course evaluations
5.3.2.7 Course certificates to indicate successful completion of training
5.3.3 The committee may also be responsible for making lodging, transportation, and meal
arrangements for course participants.
5.3.4 It is recommended that training be carried out using E. coli or Salmonella unless another
PulseNet organism is specifically requested.
5.3.5 Refer to PND02 (SOP for TIFF Image Analysis) and PND03 (SOP for Accessing and
Using the PulseNet Listserv) in the preparation of materials. Host labs can also contact
the CDC PulseNet Database Team for training materials.
5.3.6 Refer to Appendix PND10-01 for recommended training topics, and to PND01 (SOP for
Computer Equipment and Supplies) for a list of necessary materials.
5.3.7 Assign training responsibilities to trainers.
5.3.8 It is recommended that there be one trainer per five participants and a maximum of two
participants per computer.
5.3.9 At the completion of training, participants should fill out evaluations and be awarded
certificates of completion.
5.3.10 Summarize evaluations and supply trainers and members of the agenda committee a
summary of the training evaluations.

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:

8.1 PulseNet Computer support CDC contact: Brenda Brown

(404) 639-3942
[email protected]

8.2 Training support at CDC: PulseNet Database Administration Team

(404) 639-4558
[email protected]

9. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 3
 STANDARD OPERATING PROCEDURE FOR BIONUMERICS SOFTWARE CODE: PND10
Effective Date:
TRAINING 5 09 2005

Appendix PND10-01

Sample BioNumerics and PulseNet Masterscripts Training Course Agenda

SAMPLE AGENDA
Beginning BioNumerics Workshop for PulseNet Participants

General Information about the Workshop

Welcome and Introductions; Overview of Workshop

Overview of BioNumerics—Goals and Objectives

BioNumerics 3.0/3.5 and PulseNet Master Scripts

Beginning BioNumerics: Processing PFGE Gel Images

Exercise 1: Processing a PFGE gel image and linking entries to a database

Review of Morning Sessions: Questions and Answers

How to Setup Your SecurID Key Fob, Authenticating to CDC Firewall, and Using the
PulseNet BioNumerics Online Server

Uploading and Accessing Data via the National Server

Exercise 2: Uploading data to the National server

Questions and Answers

Exercise 3: Practice using Salmonella or E. coli clients to analyze and enter data

Review of Day 1: Questions and Answers

Basics behind Comparisons and Clustering

Creating Comparisons in BioNumerics

Exercise 4: Performing comparisons

Communication with PulseNet and WebBoard

Exercise 5: Pulling it all together: practice scenario

Questions and Answers, Return Evaluation Form,

Presentation of Certificates

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 3
 STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS CODE: PND11
Effective Date:
MASTERSCRIPTS FOR NEW PULSENET DATABASES 9 24 2007

1. PURPOSE: To describe the procedure for beta testing BioNumerics MasterScripts for new PulseNet
databases.

2. SCOPE: This procedure applies to CDC staff and PulseNet laboratories outside CDC who participate
in beta testing exercises.

3. DEFINITIONS/TERMS:

3.1. APHL: Association of Public Health Laboratories

3.2. SOP: Standard Operating Procedure
3.3. CDC: Centers for Disease Control and Prevention
3.4. TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics.
3.5. Bundle file: A file generated within BioNumerics for sharing and comparing PFGE patterns and
associated demographic and laboratory data from one database to another. The file extension is .bdl
and is located in the bundle folder of the corresponding database directory (X://Program
files/bionumerics/data/test_organism database/bundle)
3.6. BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths (Sint-Martens-
Latem, Belgium.
3.7. Scripts: Macros (computer commands) which allow a repeated task in BioNumerics to be
automated.
3.8. MasterScripts: Macros designed specifically for a PulseNet laboratory to submit and query PFGE
patterns and demographic data on a national database.
3.9. Database Team Members: personnel at CDC who have been designated to manage a particular
PulseNet database; these people manage all administrative aspects of the databases, including being
involved in script creation, testing, naming patterns, and correcting band markings.
3.10. On-line: Connected to the PulseNet server. Certified individuals may access the information within
the particular on-line database.
3.11. Test database: Term used to describe the new database that has been created in which to test the
MasterScripts for the new database.
3.12. Beta testing: Testing a pre-release version of the MasterScripts by making it available to selected
users.
3.13. Screen dump: By pressing the “print screen” key on a computer keyboard, a user captures an image
of whatever is currently displayed on the monitor. A user may then paste this image into another
program such as Microsoft PowerPoint.
3.14. PFGE inbox: An email account that is maintained and checked by all database team members at
CDC. The address is [email protected].
3.15. Remote testing lab: A PulseNet-participating lab other than CDC that has been designated to
perform testing on new scripts.

4. RESPONSIBILITIES AND PROCEDURE:

4.1. Internal Testing to be completed at CDC by Database Team Member(s).

4.1.1. The Database Team Member(s) will use the Testing Results_Client and Testing Results
_Admin spreadsheets (excel files found in P:\BioNumerics\Scripts\Testing\Testing
Templates) as guidelines to complete the beta testing and record any errors or
comments as necessary. Any errors should be captured in a screen dump and explained
in a PowerPoint slide.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 7
 STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS CODE: PND11
Effective Date:
MASTERSCRIPTS FOR NEW PULSENET DATABASES 9 24 2007

4.1.2. The Testing Results_Client and Testing Results_Admin spreadsheets and the
PowerPoint slides will be saved in P:\BioNumerics\Scripts\Testing\MasterScripts v##
testing and/or Organism Testing.
4.1.3. The Database Team Member(s) will notify the PulseNet Database Unit Chief and
PulseNet Computer Support once testing has been completed and the spreadsheets and
PowerPoint slides are ready for review.
4.1.4. PulseNet Computer Support will review the spreadsheets and address any script errors
or comments as necessary and fill in any corrective measures taken. Once this is
completed, the Database Team Member and Database Unit Chief are notified for
additional testing as necessary.
4.2. External testing is to be completed by a remote testing lab. This can be a laboratorian from a state
or local health department who has been analysis-certified by CDC for at least one organism (see
Certification SOP PNQ02).
4.3. At least one PulseNet participant from a remote testing lab and a Database Team Member should
beta test the MasterScripts before new MasterScripts are distributed to PulseNet participants and the
new database is considered “on-line” (if a new database was added to the MasterScripts) and
accessible to analysis-certified personnel.
4.4. Those performing testing at CDC will need to have login access to the test database. This is
accomplished by:
4.4.1. The Database Team Member(s) responsible for the test database initiates an e-mail to
PulseNet Computer Support or other responsible individual for request to access the on-line
test database for beta testing of new scripts.
4.4.2. PulseNet Computer Support will initiate access to the test database.
4.4.3. Login and password information will be given to the individual(s) who will be completing the
beta testing once approval for access has been granted.
4.5. The Database Unit Chief will forward all necessary documents and files needed for beta testing to
the remote testing lab. These documents and files include:
4.5.1. Cover letter (see Appendix PND11-1)
4.5.2. Instructions for setting up a new database in BioNumerics (taken from BioNumerics manual
section “Setup”)
4.5.3. Instructions for installing new scripts from the MasterScripts CD
4.5.4. CD containing the newest beta version of the MasterScripts
4.5.5. File(s) of testing PFGE gel(s) (.tif)
4.5.5.1. PFGE gel file name is BETA TEST GEL_organism.tif
4.5.6. PFGE report containing necessary demographic and laboratory data for testing PFGE gel(s)
4.5.7. Checklist for beta testing new MasterScripts (see Appendix PND11-2)
4.6. PulseNet participants in the remote testing lab should read and become familiar with instructions
for creating a new database and installing scripts prior to the start of beta testing.
4.7. The following procedure for beta testing scripts for a new PulseNet national database is as follows:
4.7.1. Follow instructions to create a new PulseNet database in BioNumerics. The name of the
database should be test_organism initials (i.e. test_EC).
4.7.2. Follow instructions to install MasterScripts from CD in BioNumerics
4.7.3. Analyze the test PFGE gel(s) in BioNumerics
4.7.4. Link lanes and enter demographic and laboratory data as described in the PFGE report(s)
4.7.5. Create a bundle file (.bdl) and e-mail it to the PFGE inbox with “Test Bundle” in the subject
line of the email.
4.7.5.1. This step ensures that the reference standard and entry property fields have been
installed correctly in the client database.
4.7.6. Once the bundle file has been sent, await confirmation from the PulseNet Database Team
VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 7
STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS CODE: PND11
Effective Date:
MASTERSCRIPTS FOR NEW PULSENET DATABASES 9 24 2007

4.7.7. Create a comparison of all entries in the local database

4.7.8. Select the experiment type icon (e.g. PFGE-XbaI) on the bottom of the comparison window
to visualize the PFGE patterns. Note: All patterns should be visible and no remapping errors
should be shown.
4.7.9. Perform queries on your local test organism database. Make note of any errors or anything
that does not perform correctly.
4.7.10. If a new database has been added to the MasterScripts, look over the fields, pick lists, and
organization of the organism database. Make note of anything that appears incorrect and/or
list any ideas you have to improve the look or organization of the new database.
4.7.11. Perform any other queries or actions as directed by the PulseNet Database Team. This will
include any new items or tools added to the MasterScripts.
4.7.12. The checklist (Appendix PND11-2) should be completed and comments added and sent back
to the PFGE inbox.
4.7.13. The Database Team Member responsible for beta testing of the database should give
feedback and results to all participants involved with beta testing of MasterScripts within 2
weeks of the testing date.
4.7.14. If errors are found, the PulseNet Database Team will communicate those errors to PulseNet
Computer Support or Applied Maths for resolution.
4.7.15. Once all testing is successfully completed, the MasterScripts can be distributed and any new
databases should then become accessible on-line to all analysis-certified participants.
4.7.16. The certification process should be initiated for PulseNet laboratories interested in submitting
data.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 7
 STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS CODE: PND11
Effective Date:
MASTERSCRIPTS FOR NEW PULSENET DATABASES 9 24 2007

5. FLOW CHART:

Beta Testing MasterScripts

for New PulseNet
Databases External Testing of
Client MasterScripts

Database Unit Chief asks various

Internal Testing of labs to test MasterScripts
Client and Admin
MasterScripts

Database Unit Chief sends a

cover letter, instructions, the CD
containing MasterScripts, testing
CDC Database Team files (TIFF), and a checklist to
Member(s) test MasterScripts, external testing labs
complete Testing Results
spreadsheets, and create
necessary PowerPoint slides to
show any errors External labs follow the instructions for
installing and testing the new
MasterScripts. Once testing is
completed, the lab sends the checklist
with any errors and/or comments back
to CDC
CDC Database Team
Member(s) notify Database
Unit Chief and PulseNet
Computer Support once
testing is completed
PulseNet Computer Support
and the Database Team
Member(s) review the
checklists and address any
errors and/or comments
PulseNet Computer Support
reviews Testing Results
spreadsheets and addresses any
errors and notifies Database Unit
Chief and Database Team External testing is complete
Member(s) if additional testing is once all errors have been
necessary addressed

The new MasterScripts are

distributed to participating labs
Internal Testing is complete
once all errors have been
addressed
If applicable, new database
becomes accessible on-line
and certification sets are made
available

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 7
 STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS CODE: PND11
Effective Date:
MASTERSCRIPTS FOR NEW PULSENET DATABASES 9 24 2007

6. BIBLIOGRAPHY:

7. CONTACTS:

7.1 CDC PulseNet Computer Support

Brenda Brown
(404) 639-3942
[email protected]

7.2 CDC PulseNet Database Team

(404) 639-4558
[email protected]

7.3 PulseNet Database Team Unit Chief

Kelley Hise
(404) 639-0704
[email protected]

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 7
 STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS CODE: PND11
Effective Date:
MASTERSCRIPTS FOR NEW PULSENET DATABASES 9 24 2007

Appendix PND11-1

Beta Testing MasterScripts Cover Letter Template

Beta testing organism MasterScripts vXX Beta

Scripts for PulseNet participating laboratories enable users to share and compare patterns within the
PulseNet community. Reference systems (H9812), pick lists, and other functions within BioNumerics are
standardized using scripts so that data is comparable among participating laboratories. These scripts are
developed by CDC and Applied Maths and are highly customized for each national database.

Before a set of scripts are implemented for use by the PulseNet community, a series of beta testing is done
at both CDC and remote PulseNet laboratories. These testing procedures test the functionality of the
database (i.e. ability to analyze and enter demographic/laboratory data, queries, etc.) as well as the overall
layout and look of the database. This testing is greatly needed and participation from PulseNet
laboratories is always appreciated.

This packet contains all the necessary information for remote PulseNet laboratories to beta test scripts for
new PulseNet national databases. This beta testing will be for the organism national database which is
considered online and in the production phase of testing. If at any time you have questions, please do not
hesitate to call or e-mail the PulseNet Database Team at 404-639-4558 or [email protected].

Below are the following documents and CD that are included in this packet:

1. CD containing 2 folders:
a. MasterScripts vXX Beta: has the files for testing organism scripts
b. Testing: has PFGE TIFFs of organism and TIFF demographic information, electronic copy of
checklist and procedures
2. Laboratory and demographic data for PFGE TIFFs
3. Checklist for testing the MasterScripts for the organism database

Thank you again for testing the MasterScripts vXX Beta. Your participation in PulseNet and in the testing
has been greatly appreciated.

PulseNet Database Team

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 7
 STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS CODE: PND11
Effective Date:
MASTERSCRIPTS FOR NEW PULSENET DATABASES 9 24 2007

Appendix PND11-2

Checklist for testing MasterScripts vXX Beta for PulseNet

organism database

LabID____________ Date Testing Completed_______________

Insert an “X” Item to be Tested for organism

when
completed
Create a local test organism database

Install MasterScripts vXX Beta for the test organism database

Analyze a organism PFGE gel (test of gel analysis)

Add demographic information for gel (test of pick lists, etc)

Create bundle file and e-mail to: [email protected] with “Test Bundle” in subject line (test of
bundle file function)

Perform queries on your local test organism database

Create comparison and pull up patterns to view in comparison window

Look over the fields, pick lists and organization of the organism database (please comment
below)

Comments:

After completing, either fax [404-639-3333 (ATTN: PulseNet Database Team)] or email to
[email protected] and put “Testing” in the subject line. Please don’t forget to fill in the LabID information at
the top.

Thank you.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 7
 STANDARD OPERATING PROCEDURE FOR NAMING PULSENET OUTBREAKS CODE: PND12
Effective Date:
AND CLUSTERS 4 01 2006

1. PURPOSE: To describe the PulseNet standardized nomenclature for outbreaks and clusters.

2. SCOPE: This procedure applies to all CDC PulseNet Database personnel entering outbreak and cluster
information into the “Outbreak” field in BioNumerics.

3. DEFINITIONS/TERMS:

3.1 TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics.
3.2 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
3.3 CDC: Centers for Disease Control and Prevention
3.4 PFGE: Pulsed-field Gel Electrophoresis
3.5 Cluster: A group of isolates with the same serotype determined to possess indistinguishable PFGE DNA
patterns using one or more enzyme restrictions.
3.6 DNA: Deoxyribonucleic acid
3.7 LITS: Laboratory Information Tracking System
3.8 LabID: Unique identification assigned to each PulseNet participating laboratory, usually two-four letters
that correspond with the postal code that is assigned by CDC.
3.9 PulseNet Listserv: A closed, moderated, electronic web conference used for communication among
PulseNet participants. The PulseNet Listserv is open to all laboratory staff at PulseNet participating
laboratories. Epidemiologists at these laboratories, when approved by the PulseNet laboratory contact,
U.S. food regulatory staff, and PulseNet International representatives may also have access. The PulseNet
Listserv is not open to university or private industry personnel unless their inclusion is deemed to be in the
interest of public health.
3.10 Outbreak: A cluster of cases of infections with a common epidemiological exposure, e.g. to a specific food
product.
3.11 Cluster: A group of isolates with the same serotype with indistinguishable PFGE DNA patterns by one or
more restriction enzyme

4. RESPONSIBILITIES/PROCEDURE:

4.1 Basic code: A code consisting of the date of recognition of the cluster in the YYMM (2-digit year and 2-
digit month) format is created, followed by the LabID abbreviation where it was first recognized (via
PulseNet Listserv or other such posting).
<Date><LabID><LITS code for organism>-<number of cluster in month>
4.1.1 If a cluster is recognized at more than one lab simultaneously, the letter code should be “ml” for
multi location.
4.1.2 The reason for this order of characters in the basic code is so it will be possible to query and sort
the result of a query by year using only this single field.
4.2 The first characters identify the cluster/outbreak in a unique way. A dash (“-”) plus a digit should follow
the initial 9-11 characters (accounting for LabIDs with >2 characters) to indicate:
4.2.1 The number of the cluster/outbreak for the month. There can be different clusters/outbreaks with
different serotypes occurring simultaneously in the same state; hence, to avoid the risk of mixing
information on separate clusters/outbreaks together in the databases, the three-letter code for the
serotype used in the naming of the PFGE-profiles should also be added after the date information.
4.2.1.1 Example: 0805MAJEG-2 is the second Salmonella Enteritidis outbreak in Massachusetts, first
seen in May 2008. “0805” indicates the cluster was first noticed in May of 2008; “MA”
indicates that it was first seen in MA; “JEG” represents Salmonella Enteritidis; the “-2”
indicates that this is the second outbreak of this serotype for this month recognized in MA.
4.2.2 A “?” indicating that the status of the profile/case has not been firmly established as being
associated with the cluster
4.2.3 An “x” indicating isolates that are being used as controls in a study
4.2.4 Examples:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 2
 STANDARD OPERATING PROCEDURE FOR NAMING PULSENET OUTBREAKS CODE: PND12
Effective Date:
AND CLUSTERS 4 01 2006

4.2.4.1 0805mlGX6-1 (the first Listeria monocytogenes multi-state outbreak recognized

simultaneously in two or more states in May 2008)
4.2.4.2 0210MSJPX-3 (a cluster of cases associated with Salmonella Typhimurium discovered in
October 2002 following two other clusters/outbreaks in Mississippi that month
4.2.4.3 0207FLJ16-2? The second Shigella sonnei Florida outbreak turns out to be a big one and
there are a lot of isolates that may be related to the outbreak. It takes time to sort out whether
they are part of the outbreak (the database manager has received a lot of TIFFs or they have
done a search on the profile but haven’t analyzed the results, yet). It could also be a single
unconfirmed isolate from a patient in a different state in a presumably single state outbreak.
4.2.4.4 0309WADBR-1? This is a big one, recognized as a cluster of Campylobacter jejuni in
Washington in September of 2003. Before they get interrupted, the database manager names
all the possible matches they have found so that they may find them again easily.
4.2.4.5 0307GAJJP-1x This is the first outbreak of Salmonella Newport that began in July of 2003 in
Georgia; however, the “x” on the end denotes that these were considered to be controls in the
outbreak, and do not have the outbreak pattern. This simplifies being able to pull these up at a
later date.

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:
8.1 2009-01-01: Removed the “c” that used to be placed at the end of the basic outbreak code indicating the
cluster was not yet an outbreak. Also removed the “ml” that used to be placed at the end of the basic outbreak
code indicating the cluster was a multi-state cluster.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 2
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

1. PURPOSE: to describe the standardized protocol for analysis of MLVA data of Shiga toxin-
producing Escherichia coli O157 (STEC O157) and Salmonella enterica serotypes
Typhimurium and Enteritidis in BioNumerics.

2. SCOPE: to provide PulseNet participants with a single protocol for analyzing MLVA data of
STEC O157 and Salmonella serotypes Typhimurium and Enteritidis, thus ensuring inter-
laboratory comparability of the generated results.

3. DEFINITIONS:
3.1.MLVA: Multiple-locus variable-number tandem repeat analysis
3.2.VNTR: Variable-number tandem repeat
3.3.CDC: Centers for Disease Control and Prevention
3.4.SOP: Standard Operating Procedure

4. RESPONSIBILITIES/PROCEDURE:
4.1 Software needed for data analysis
4.1.1. BioNumerics version 4.5 or higher
4.1.2. Customized scripts for data import (VNTRImport_v4.bns), copy number
calculation (= allele assignment) (VNTRCalc_v4.bns), troubleshooting
(VNTRReport.bns) and support functions (VNTRDetails_v2.bns).
4.1.3 Look-up tables (BeckmanEcoli.txt, BeckmanST.txt, BeckmanSE.txt) for allele size
ranges
4.2 General overview
4.2.1 The analysis process consists of three major steps:
4.2.1.1 Exporting the appropriate data file from the Beckman system (refer to SOP
PNL19, step 4.10.1.6)
4.2.1.2 Importing this file into BioNumerics (using the script VNTRImport_v4.bns)
4.2.1.3 Determining the copy numbers (assigning alleles) for each VNTR (using
the script VNTRCalc_v4.bns and the organism-specific look-up table
BeckmanEcoli.txt, BeckmanST.txt, BeckmanSE.txt)
4.2.1.3.1 VNTRCalc_v4.bns script facilitates the allele assignment in two
different ways:
4.2.1.3.1.1 “BeckmanEcoli, BeckmanST and BeckmanSE” – assign the
copy number based on the look-up table (PND14-1a-c). This
method should be used in all routine analysis.
4.2.1.3.1.2 “Predicted” – calculates the copy number based on the
mathematical formula: (Observed fragment size –
offset)/repeat size. This method should only be used when a
new allele (not specified in the look-up table) is identified
4.3 Required import format
4.3.1 The data should be exported from the Beckman CEQ/GeXP software as CSV
(comma-delimited) text files containing the fragment length information.
Multiple columns will be present in the file but only four columns are of interest:
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

“RN,” “dye,” “est size (nt)” and “pk height (rfu)”. All other columns will be
ignored by the script during import. The field “RN” should contain information
designating the BioNumerics key number (isolate identifier) loaded in each well
of the sequencer plate run, as well as which VNTR mastermix or reaction was
loaded. The import script will ignore any text that appears beyond the dot “.” in
the RN field.
4.3.1.1 NOTE: no spaces or underscores are allowed between the strain ID and the
reaction ID or between the reaction ID and the dot “.” in the RN field.
4.4 Setting up a new database
4.4.1 Click “Create New Database” icon to set up a new database
4.4.2 Type in the name (for example “O157MLVA”) of your new database and click
”Next”
4.4.3 Select the directory (default on CDC training laptops is
[HOMEDIR]\O157MLVA; this will save the data on the G-drive) in which you
want to set up the new database. Choose the default “Yes(recommended)” when
asked “Do you want to automatically create the required directories?”. Click
“Next”.
4.4.4 Choose “Yes” to enable creation of log files and click “Finish”
4.4.5 In the appearing “Set up new database” pop-up window, choose “Local database
(single user only)” and click “Proceed”
4.4.6 Click “Yes” in the next pop-up confirmation window (“Choosing ‘local database’
will restrict some functionality of the software. Are you sure you want to
continue?”) and “Proceed” in the following “Plug-in” pop-up window
4.4.7 Close the newly created database.
4.4.8 Go to the directory in which you placed your database at step 4.4.3 (on CDC
training laptops: C:\Program files → BioNumerics → Data), open folder
“O157MLVA” and create two subfolders named “Scripts” and “VNTRtables”.
4.4.9 Save the four MLVA specific scripts (VNTRImport_v4.bns, VNTRCalc_v4.bns,
VNTRReport.bns, VNTRDetails_v2.bns) in the “Scripts” folder and the organism
specific assay look-up table (e.g. BeckmanEcoli.txt) in the “VNTRtables” folder
4.4.10 Open the database. The scripts (except the VNTRDetails script) should now
appear under the “Scripts” drop-down menu
4.5 Importing a peak file for the first time
4.5.1 Review the peak file: make sure that the observed size for the D1 labelled
molecular size standard peaks is within ± 1 bp from the expected size. Remove
any data (failed reactions, controls, internal ladder) that you don’t want to import
in the BioNumerics from the CSV file. Re-name and re-save the CSV file either
on your hard drive or on the flash drive.
4.5.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” drop-down
menu. The “Import VNTR peak data” dialog box will appear. From the “Peak file
format” drop-down menu, select “Beckman peak file”. The script pops up a file
dialog box, prompting for the name of the file to import. Select the appropriate

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

file and click “OK”. A second dialog box pops up, prompting for a variety of
other information:
4.5.2.1 Fingerprint file name. This is the name of the fingerprint file that will be used
in BioNumerics. Leave this unchanged, unless a file has already been
imported with the same name. In this case, change the file name into a new,
unique and informative name.
4.5.2.2 Dyes to import. The first dye (D1) contains only reference markers and does
not need to be imported. Use this list to select what dyes will be imported;
they are D2, D3 and D4. Each dye will be stored in a different file, resulting
from appending the dye name to the fingerprint file name.
4.5.2.3 Assign reference positions. Leave this option checked.
4.5.2.4 Select imported isolates. Leave this option checked.
4.5.2.5 Pool tags. For this protocol, there are two pool tags representing the two PCR
reactions for each isolate (all loaded on different wells) and tagged with
names “R1” and “R2”. The two tags should appear in the list. If they do not,
they can be added by selecting “Add” to update.
4.5.2.6 If everything is filled appropriately, click “OK” to start the import.
4.5.2.7 NOTE: These settings are automatically saved and reloaded the next time this
script is run.

4.5.3 When the script is finished, it has:

4.5.3.1 Created a fingerprint type “VNTRFpr” with appropriate settings
4.5.3.2 Created fingerprint types with appropriate settings for each dye and each pool
used. The names of these fingerprint types are “VNTRFpr”+“pool
name”+“dye” (for example: VNTRFprR1_D2).
4.5.3.3 Created fingerprint files for each dye in the fingerprint type “VNTRFpr,” and
imported the band information in these lanes. The individual lanes are
assigned to their appropriate pool fingerprint type, according to the known
pool tags found in the “RN” field.
4.5.3.4 Created new database entries for all isolates found
4.5.3.5 Linked the fingerprints to their corresponding entries
4.5.3.6 Selected all isolates for which fingerprint data was imported
4.6 Determining the copy numbers for the first time (allele assignment)

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

4.6.1 Run the script VNTRCalc_v4 from the scripts menu. The first time this script is
run, it will automatically create a new character set called “VNTR_vals”. This
character set will hold the copy numbers for each VNTR. The script will also
create a second character set called “VNTR_frags” which holds the fragment size
with the highest fluorescence for each VNTR. Click “OK” on the two pop-up
windows notifying that these two character types will be created.
4.6.2 Before the script can determine copy numbers from the fragment length
information, it should have sufficient information about what VNTRs can be
found in which pool and dye, what are the repeat lengths, etc. To this end, the
script pops up a dialog box called “VNTR assign”. This dialog box contains a list
of defined VNTRs that is initially empty. To enter the parameters of the first
VNTR, click on “Add”. This brings up a second dialog box, prompting for all
properties of this first VNTR. Refer to the appendices PND14-2a-c for all
necessary information to load the specifications for each VNTR.

4.6.3 The VNTR information includes:

4.6.3.1 Name. This name will be used for further reference and for the character name
in the character type VNTR_vals. Each VNTR should have a unique name (for
example VNTR_3, VNTR_34, etc).
4.6.3.2 Offset. Each fragment consists of a repeat portion and a constant portion, due
to the fact that the primers do not occur exactly at the start and the end of the
repeat region. This parameter specifies the size (in base pairs) of the constant
portion.
4.6.3.3 Repeat length. This parameter specifies the size of a unit repeat block (in base
pairs).
4.6.3.4 Copy range. These parameters specify the minimum and maximum number of
copies that the script will consider during the copy number determination.
4.6.3.5 Tolerance. This specifies the maximum difference between the expected
fragment length (calculated by the software) and the actual length estimated
by the sequencer.
4.6.3.6 Take from fingerprint type. This drop-down list should be used to indicate on
what fingerprint type this VNTR was run. The fingerprint type is determined
by the dye and the pool tag.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

4.6.4 If all parameters are filled in appropriately, click OK to add the VNTR and return
to the previous dialog box (“VNTR assign”). Repeat the same actions to enter the
information for all VNTRs used for this protocol. When all VNTRs are entered,
make sure the box for “Code absent as negative” is checked. Select the
appropriate assay specific look-up table (for example “BeckmanEcoli” for STEC
O157) from the “Fragment ranges” drop-down menu and press the
“Save&Assign” button to let the script assign copy numbers for the currently
selected entries.
4.6.4.1 NOTE 1: pressing “Save&Quit” would store the information without
calculating copy numbers for the current selection.
4.6.4.2 NOTE 2: all information regarding the VNTRs is stored with the database.
The next time the script is run, the VNTR definitions will be loaded
automatically.
4.6.4.3 For every fragment with the highest fluorescence level in the fingerprint type
(= the combination of the reaction and the dye), the script will assign an allele
type (=a copy number) based on the fragment size ranges specified in the
appropriate look-up table (appendices PND14-1a-c). Note that, within the
same fingerprint type, more than one VNTR can be loaded if there is no
overlap within the fragment size ranges.
4.6.5 When the script has completed, all copy numbers for all VNTRs for the currently
selected entries are determined.
4.6.5.1 Note: Two types of problems may arise during the process (if one or more
such errors were encountered during the calculations, an error report is
displayed listing all the problems):
4.6.5.1.1 None of the peaks present in a fingerprint are compatible with the
fragment size ranges in the look-up table. In this case, the
corresponding character value will be scored “-2.0”. In this situation,
run the “VNTRReport” script from the “Scripts” drop-down menu to
verify the reason for the problem:
4.6.5.1.1.1 No amplification (a null allele): mutations, insertions or deletions
in the primer annealing region, or locus located on a plasmid that
was lost. PCR needs to be repeated only if the null allele occurred
in a locus in which null alleles have not been previously detected
(For STEC O157: VNTR34, VNTR25, VNTR17; and in the case
of VNTR19 for which null alleles are extremely rare, and hence
should always be confirmed. For Salmonella serotype
Typhimurium: ST2, ST3, ST6, ST7 and in the case of ST8 for
which null alleles are extremely rare, and hence should always be
confirmed. For Salmonella serotype Enteritidis: VNTR5, VNTR8,
VNTR9; and in the case of SE6 for which null alleles are
extremely rare and hence always need to be confirmed).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

4.6.5.1.1.2 Fragment size outside the acceptable range (either slightly outside
of range for a previously detected allele or a possible new allele).
Rerun the fragment analysis reaction to confirm the accuracy and
reproducibility of the sizing. The isolate should be submitted to
CDC for confirmation only if a possible new allele is detected. If
the sizing is reproducibly slightly outside the range for an existing
allele, the CDC database managers will adjust the look-up table
based on the evidence the submitting laboratory provides.
4.6.5.1.2 More than one peak in a fingerprint is compatible with an acceptable
fragment size range. In this case, the script will use the solution that
corresponds to the peak with the largest “peak height” value. Possible
causes for multiple peaks:
4.6.5.1.2.1 Primer stutter: multiple peaks with sizes 1-2 bp from each other.
No further action required.
4.6.5.1.2.2 Double alleles: two peaks differing by one or more full repeats
from each other. Further actions required for all other loci except
Salmonella serotype Enteritidis locus SE8 (in a subset of strains
double alleles are known to occur and the major (larger) allele
always has the highest fluorescence):
4.6.5.1.2.2.1 Repeat the PCR with a freshly made template. If two peaks
still observed and the same peak consistently has the
highest fluorescence intensity, report the predominant peak
only. If the same peak does not consistently have the
highest fluorescence intensity, proceed to the next step.
4.6.5.1.2.2.2 Test ten single colony picks from the culture. Report the
peak that has the highest fluorescence intensity in the
majority of the picks.
4.7 Analyzing a peak file on a routine basis
4.7.1 Review the peak file: make sure that the observed size for the D1 labelled
molecular size standard peaks is within ± 1 bp from the expected size. Remove
any data (failed reactions, controls, internal ladder) that you don’t want to import
in the BioNumerics from the CSV file. Re-name and re-save the CSV file either
on your hard drive or on the flash drive.
4.7.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” drop-down
menu. The “Import VNTR peak data” dialog box will appear. From the “Peak file
format” drop-down menu, select “Beckman peak file”. The script pops up a file
dialog box, prompting for the name of the file to import. Select the appropriate
file and click “OK”. “VNTR/Import peak data” dialog box will appear. De-select
“D1” from the “Dyes to import” and click “OK”.
4.7.3 Run the script VNTRCalc_v4 from the scripts menu. “VNTR assign” dialog box
will appear. Select the appropriate look-up table (for example “BeckmanEcoli” for
STEC O157)from the “Fragment ranges” drop-down menu and click
“Save&Assign” to assign allele numbers.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

4.8 Verifying the allele assignment

4.8.1 For each isolate in the database that has some VNTR data associated, you can
click on the “VNTR frags” and “VNTR vals” entries in the list of yellow buttons
to open the “Entry edit” windows.

4.8.2 If the VNTRCalc_v4 script did not detect a fragment for a VNTR, refer to the step
4.6.5.1.1 of this protocol for details on how to proceed. If the fragment size is
slightly outside the range specified in the look-up table it is possible to manually
assign a temporary allele size and type for that VNTR until an official
confirmation has been performed by CDC.
4.8.2.1 Note: only the CDC database managers are allowed to modify the look-up
table txt-files. Once a modification has been made, the modified file will be
posted on the PulseNet SharePoint site under QA/QC manual. An automatic e-
mail notification will be sent about the change in the SOP.
4.8.2.2 To manually assign an allele size and type click on the fragment size in the
“VNTRfrags” entry edit window or the allele type in the “VNTR vals” entry
edit window. This will open the “Change character value” window in
BioNumerics lower than 5.0. In BioNumerics 5.0 the allele size or type can be
directly highlighted and changed in the entry edit window (Screen shots
below)

4.9 Creating a composite data set to visualize the allele types

4.9.1 In order to display copy numbers next to a dendrogram in a comparison, first
create a “composite data set” that holds the VNTR data.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

4.9.1.1 NOTE: The created composite dataset will be automatically saved in the
database, and hence only need to be created once before the first analysis in
the database.
4.9.1.2 From the “Experiments” drop-down menu, select the option “Create new
composite data set…”, enter a name (e.g. VNTR_cmp), and click the “OK”
button. The “Composite data set ‘VNTR_cmp’” window will appear.
4.9.1.3 Highlight the experiment VNTR_vals and from the “Experiment” drop-down
menu, select the option “Use in composite data set”. Close the window.
4.9.2 Next time a comparison window is opened (see below step 4.10), there will be a
new experiment VNTR_cmp listed in the bottom of the window (BioNumerics
versions lower than 5.0) or in the top left corner of the window (BioNumerics
version 5.0). This experiment will facilitate the display of a spreadsheet-like view
of the copy numbers (note that it may be necessary to scroll the experiment list to
the right with the arrow button to bring VNTR_cmp in display in BioNumerics
versions lower than 5.0). This can be shown next to a dendrogram analysis of the
data set.
4.10 Performing comparisons based on the VNTR data
4.10.1. The VNTR data contained in the character set VNTR_vals can be analysed in
BioNumerics with all the tools that are available for character data. That includes
cluster analysis with a variety of methods and similarity coefficients. For VNTR
data, the coefficients that make most sense are:
4.10.1.1 Categorical: preferred if differences in copy numbers should be treated in a
qualitative way. This is the only option for creating dendrograms using MLVA
data.
4.10.1.2 Manhattan: preferred if differences in copy numbers should be treated in a
quantitative way (i.e. a larger difference means more distantly related
organisms). This coefficient can be used to construct minimum spanning trees.
4.10.2 In order to create a dendrogram:
4.10.2.1 Select the isolates to be included in the dendrogram
4.10.2.2 From the “Comparison” drop-down menu, select the option “Create new
comparison” and a “Comparison” window will appear
4.10.2.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics versions
lower than 5.0 or from the top left “Experiments” window in BioNumerics
5.0.
4.10.2.4 From the “Clustering” drop-down menu, select the option “Calculate…Cluster
analysis (similarity matrix)” and a “Composite data set comparison” dialog
box will appear
4.10.2.5 Select “Categorical” for “Multi-state coefficient” and “UPGMA” for
“Dendrogram type”
4.10.2.6 Click on “OK” button to finish the calculations and the “Comparison” window
with the dendrogram will reappear
4.10.2.7 From the “Layout” drop-down menu, select the option “Show image”

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

4.10.2.8 From the “Composite” drop-down menu, select the option ”Show
quantification (values)” and the copy numbers will appear next to the
dendrogram

4.10.3 In order to create a minimum spanning tree:

4.10.3.1 Select the isolates to be included in the spanning tree
4.10.3.2 From the “Comparison” drop-down menu, select the option “Create new
comparison” and a “Comparison” window will appear
4.10.3.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.3.4 From the “Clustering” drop-down menu, select the option
“Calculate…Minimum spanning tree (population modeling)” and a
“Minimum spanning tree” dialog box will appear
4.10.3.5 Make sure the default “Manhattan” is checked for “Coefficient” and click
“OK” and the “Minimum spanning tree” window with the tree will appear.
4.10.3.6 You can find out the content for each node by clicking on them individually.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:
7.1. Eija Trees, D.V.M., Ph.D.
PulseNet Next Generation Subtyping Unit, EDLB, DFWED, CDC
(404) 639-3672
[email protected]
7.2. Patti Lafon
PulseNet Next Generation Subtyping Unit, EDLB, DFWED, CDC
(404) 639-2828
[email protected]
7.3. Ashley Sabol
PulseNet Next Generation Subtyping Unit, EDLB, DFWED, CDC
(404) 639-2947
[email protected]

8. AMENDMENTS:
1/27/2009: the allele (= copy number) assignment will be based on a look-up table listing the
actual observed fragment size ranges instead of using the mathematical formula that assigns
alleles based on predicted fragment sizes. The look-up table approach in allele assignment will
facilitate the comparison of fragment size data from the Beckman Coulter CEQ 8000/8800/GeXP
and the Applied Biosystems Genetic Analyzer 3130xl in the same database.
4/8/2013: appendix PND14-2 (BioNumerics specifications for the STEC O157 VNTR loci) was
added.
4/8/2013: Additions to step 4.6.5: more clarification for reasons for error messages that occur
upon allele assignment and guidance for further follow-up for these errors.
2/26/2014: Merged the SOPs for STEC O157 (PND14), Salmonella serotypes Typhimurium
(PND15) and Enteritidis (PND19) Analysis for Beckman Coulter CEQ8000/8800/GeXP Data
into one protocol.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

Appendix PND14-1a
STEC O157 VNTR Allele List and Corresponding Observed Fragment Sizes in
the Beckman Coulter CEQ 8000/8800/GeXP (BeckmanEcoli look-up table)

Note: This is posted on the PulseNet SharePoint site under the QA/QC manual as a TXT-file so users
may save it locally and use it with the BioNumerics MLVA scripts. Every time the table is updated, the latest
version is posted on SharePoint site.

Count VNTR_3 VNTR_34 VNTR_9 VNTR_25 VNTR_17 VNTR_19 VNTR_36 VNTR_37

1 471-472 116-119
2 333-334 122-124 130-133 282-284
3 339-340 153-154 482-484 128-131 137-140 291-292 123-125 159-161
4 345-346 170-172 488-490 134-136 143-146 296-299 130-132 164-166
5 350-353 188-190 494-497 141-143 149-152 302-304 136-139 170-173
6 356-359 206-209 499-503 146-148 156-159 307-311 143-146 176-179
7 362-365 224-227 506-509 153-154 162-165 314-316 150-153 181-185
8 368-371 241-245 512-515 158-160 169-172 320-322 158-161 187-191
9 373-377 260-262 518-521 165-166 175-178 325-328 165-168 194-197
10 380-383 278-280 524-528 170-171 182-184 332-334 172-175 200-203
11 386-389 295-298 530-534 188-190 337-339 179-182 207-209
12 392-396 313-314 536-540 195-197 343-345 186-189 213-215
13 398-401 543-546 188-190 199-201 349-351 193-196 219-221
14 404-407 549-552 356-357 200-202 225-227
15 410-413 555-558 208-209 361-363 207-210 231-233
16 417-419 561-564 214-216 237-238
17 423-425 566-570 222-223 241-244
18 429-431 572-576 227-228 228-230 249-251
19 435-437 579-582 235-237 255-256
20 442-444 584-587 242-243 261-262
21 448-449 590-594 246-247 250-255
22 454-456 597-599 273-274
23 460-464 603-605
24 466-468 608-611
25 473-474 614-615
26 476-477

VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

Appendix PND14-1b
S. enterica serotype Typhimurium VNTR Allele List and Corresponding Observed
Fragment Sizes in the Beckman Coulter CEQ 8000/8800/GeXP (BeckmanST look-up table)

Note: This is posted on the PulseNet SharePoint site under the QA/QC manual as a TXT-file so users may
save it locally and use it with the BioNumerics MLVA scripts. Every time the table is updated, the latest
version is posted on the SharePoint site.

Count ST3 ST5 ST7 ST10 ST2 ST6 ST8

0 113-115
1 124-126 202-203
2 164-171 146-149 130-136 322-323 207-209
3 151-153 139-145 213-215
4 174-182 162-164 151-154 335-336 219-221
5 187-191 168-170 162-164 341-343 225-227
6 172-176 172-173 347-349 231-233
7 179-182 178-183 354-357 234-239
8 186-188 187-191 360-362 167-168 242-245
9 191-194 200-201 366-369 247-250 294-296
10 196-199 209-210 373-375 254-257
11 203-205 379-381 207-209 259-263 326-328
12 208-211 218-219 264-268
13 214-217 227-229 385-388 272-274
14 220-223 232-237 392-394 248-252 278-280
15 226-228 246-247 398-400 284-287 359-360
16 232-234 255-256 404-406 264-266 290-292 373-375
17 238-239 264-265 411-413 279-281 296-298 384-385
18 243-245 273-274 417-419 289-292 302-303
19 249-251 282-284 424-426 312-316 305-310 399-40
20 255-257 291-293 430-433 319-326 314-315 417-419
21 260-262 301-302 437-439 341-343 320-322 425-426
22 266-268 443-446 351-357 325-327 432-435
23 272-274 319-322 450-452 358-363 331-333 448-452
24 278-279 456-459 365-369 336-339 460-464
25 284-286 463-465 387-392 343-344 471-475
26 290-292 397-407 349-350 485-488
27 470-472 355-357 493-497
28 476-479 425-427 361-362 501-508
29 312-314 483-485 433-438 367-368 512-513
30 489-492 373-374 526-531
31 496-498 532-537
32 503-504 385-386 547-552
VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

33 509-511 553-558
34 516-518 560-565
35 523-526 580-584
36 530-532 587-592
37 599-609
38 610-618
39 622-624
40 551-552 631-636

VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

Appendix PND14-1c

S. enterica serotype Enteritidis VNTR Allele List and Corresponding Observed

Fragment Sizes in the Beckman Coulter CEQ 8000/8800/GeXP
(BeckmanSE look-up table)

Note: This is posted on the PulseNet SharePoint site under QA/QC manual as a TXT-file so users may
save it locally and use it with the BioNumerics MLVA scripts. Every time the table is updated, a latest
version of the look-up table is posted on the SharePoint.

Count SE1 SE2 SE8 SE6 SE9 SE3 SE5

1 344-347 175-176 162-164
2 166-168 433-435 183-186
3 172-174 295-296 192-195 197-201 171-172
4 179-182 299-303 199-200 209-214 177-179
5 185-189 305-309 224-225 183-185
6 193-196 312-316 189-191
7 200-202 318-322 195-197
8 207-209 325-329 410-413 258-259 201-203
9 214-217 332-335 443-445 207-209
10 222-223 338-342 476-481 280-282 213-215
11 229-230 344-349 219-221
12 236-237 353-354 534-535 225-227
13 244-245 360-365 569-570 231-233
14 593-594 237-239
15 256-257 372-374 243-245
16 249-250
17 270-271 255-257
18 260-263
19 266-268
20 272-274
21
22 284-285
23
24 317-318
24
25
26
27
28
29
30 361-362
VERSION: REPLACED BY: AUTHORIZED BY:

Page 14 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

Appendix PND14-2a
BioNumerics specifications for STEC O157 VNTR loci

Reaction R1 R2
Locus VNTR_3 VNTR_17
Dye D2 D2
Offset 321 120
Repeat length 6 6
Copy range 0-25 0-30
Tolerance 2 2
Fragment size in EDL933 373-377 156-159
Copy number in EDL933 9 6

Locus VNTR_34 VNTR_19

Dye Quas 670 (D4) Quas 670 (D4)
Offset 100 272
Repeat length 18 6
Copy range 0-20 0-25
Tolerance 3 2
Fragment size in EDL933 278-280 307-311
Copy number in EDL933 10 6

Locus VNTR_9 VNTR_36

Dye Quas 670 (D4) Quas 670 (D4)
Offset 465 102
Repeat length 6 7
Copy range 0-50 0-20
Tolerance 2 2
Fragment size in EDL933 530-534 158-161
Copy number in EDL933 11 8

Locus VNTR_25 VNTR_37

Dye Quas 705 (D3) Quas 705 (D3)
Offset 110 142
Repeat length 6 6
Copy range 0-20 0-25
Tolerance 2 2
Fragment size in EDL933 134-136 187-191
Copy number in EDL933 4 8

Fragment size ranges based on independent runs on multiple instruments at

CDC and PulseNet Participating Laboratories

VERSION: REPLACED BY: AUTHORIZED BY:

Page 15 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

Appendix PND14-2b
BioNumerics specifications for S. enterica serotype Typhimurium VNTR loci

Reaction R1 R2
Locus ST3 ST2
Dye Quas705 (D3) Quas670 (D4)
Offset 156 62
Repeat length 6* 13**
Copy range 0-20 0-40
Tolerance 2 3
Fragment size in LT2 174-182 358-363
Copy number in LT2 4* 23**

Locus ST5 ST6

Dye Quas670 (D4) D2
Offset 139 191
Repeat length 6 6
Copy range 0-60 0-50
Tolerance 2 2
Fragment size in LT2 220-223 264-268
Copy number in LT2 14 12

Locus ST7 ST8

Dye D2 Quas705 (D3)
Offset 115 196
Repeat length 9 11***
Copy range 0-30 0-50
Tolerance 3 3
Fragment size in LT2 151-154 553-558
Copy number in LT2 4 33***

Locus ST10
Dye Quas705 (D3)
Offset 311
Repeat length 6
Copy range 0-50
Tolerance 2
Fragment size in LT2 373-375
Copy number in LT2 10

* A half of a repeat difference considered significant. Actual repeat length 12 bp and actual copy number in LT2 2.5
** A third of a repeat difference considered significant. Actual repeat length 39 bp and actual copy number in LT2 8.0
*** A third of a repeat difference considered significant. Actual repeat length 33 bp and actual copy number in LT2 11.0

Fragment size ranges are based on independent runs on multiple instruments at CDC and in PulseNet Participating
Laboratories.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 16 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF MLVA CODE: PND14
DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 (STEC O157) AND
Effective Date:
SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN 02 26 14
BIONUMERICS - BECKMAN COULTER CEQ 8000/8800/GeXP DATA

Appendix PND14-2c
BioNumerics specifications for S. enterica serotype Enteritidis VNTR loci

Reaction R1 R2
Locus SE1 SE9
Dye Quas670 (D4) Quas670 (D4)
Offset 152 166
Repeat length 7 9
Copy range 0-20 0-5
Tolerance 2 2
Fragment size in K1891 193-196 183-186
Copy number in K1891 6 2

Locus SE2 SE3

Dye D2 D2
Offset 271 165
Repeat length 7 12
Copy range 0-20 0-10
Tolerance 2 3
Fragment size in K1891 325-329 209-214
Copy number in K1891 8 4

Locus SE8 SE5

Dye Quas705 (D3) Quas705 (D3)
Offset 260 155
Repeat length 86 6
Copy range 0-3 0-25
Tolerance 4 2
Fragment size in K1891 433-435 201-203
Copy number in K1891 2 8

Locus SE6
Dye Quas670 (D4)
Offset 148
Repeat length 33
Copy range 0-15
Tolerance 3
Fragment size in K1891 476-481
Copy number in K1891 10

Fragment size ranges are based on independent runs on multiple instruments at

CDC and in PulseNet Participating Laboratories

VERSION: REPLACED BY: AUTHORIZED BY:

Page 17 of 17
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

1. PURPOSE: To describe the standardized protocol for analysis of MLVA data of

Shiga toxin-producing Escherichia coli O157 (STEC O157) and Salmonella enterica
serotypes Typhimurium and Enteritidis in BioNumerics.

2. SCOPE: To provide the PulseNet participants with a single protocol for analyzing
MLVA data of STEC O157 and Salmonella enterica serotypes Typhimurium and
Enteritidis, thus ensuring inter-laboratory comparability of the generated results.

3. DEFINITIONS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 VNTR: Variable-number tandem repeat
3.3 CDC: Centers for Disease Control and Prevention
3.4 SOP: Standard Operating Procedure

4. RESPONSIBILITIES/PROCEDURE
4.1 Software needed for data analysis
4.1.1 BioNumerics version 4.5 or higher
4.1.2 Customized scripts for data import (VNTRImport_v4.bns), copy number
calculation (= allele assignment) (VNTRCalc_v4.bns), troubleshooting
(VNTRReport.bns) and support functions (VNTRDetails_v2.bns).
4.1.3 Organism specific assay look-up tables (ABIEcoli.txt, ABIST.txt, ABISE.txt)
for allele size ranges
4.2 General overview
4.2.1 The import process consists of three major steps:
4.2.1.1 Exporting the appropriate data file from the Genetic Analyzer system
(see laboratory protocols PNL23 and PNL28 step 4.10.1.15).
4.2.1.2 Importing this file into BioNumerics (using the script
VNTRImport_v4.bns).
4.2.1.3 Determining the copy numbers (assigning alleles) for each VNTR
(using the script VNTRCalc_v4.bns and the organism specific look-up
table ABIEcoli.txt, ABIST.txt, or ABISE.txt).
4.2.1.3.1 VNTRCalc_v4.bns script facilitates the allele assignment in two
different ways:
4.2.1.3.1.1 “ABIEcoli, ABIST, and ABISE” – assign the copy
number based on the look-up tables (PND16-1a-c,
respectively).
4.2.1.3.1.2 “Predicted” – calculates the copy number based on the
mathematical formula: (Observed fragment size –
offset)/repeat size (not an option for the ABI data)

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

4.3 Required import format

4.3.1 The data should be exported from the GeneMapper software as a txt file
(tab-delimited text file) containing the fragment length information. The
following columns should appear in the exported table in the following
order from left to right: “Dye/Sample peak”, “Sample File Name”,
“Marker”, “Allele”, “Size”, “Height”, “Area”, “Data point”. The field
“Sample File Name” should contain information designating the
BioNumerics key number (isolate identifier) loaded in each lane of the
sequencer, as well as which VNTR mastermix (R1 or R2) was loaded. The
import script will ignore any text that appears beyond the dot “.” in the
“Sample File Name” field.
4.3.1.1 NOTE: no spaces or underscores are allowed between the strain ID
and the reaction ID (R1 or R2) or between the reaction ID and the dot
“.” in the RN field.
4.4 Setting up a new database
4.4.1 Click “Create New Database” icon to set up a new database.
4.4.2 Type in the name (for example “O157MLVA” for STEC O157,
“STMLVA” for Salmonella Typhimurium or “SEMLVA” for Salmonella
Enteritidis) of your new database and click ”Next”.
4.4.3 Select the directory (default on CDC training laptops is
[HOMEDIR]\O157MLVA for STEC O157; this will save the data on the C-
drive) in which you want to set up the new database. Choose the default
“Yes (recommended)” when asked “Do you want to automatically create
the required directories?” and then click “Next”.
4.4.4 Choose “Yes” to enable creation of log files and click “Finish”.
4.4.5 In the appearing “Set up new database” pop-up window, choose “Local
database (single user only)” and click “Proceed”.
4.4.6 Click “Yes” in the next pop-up confirmation window (“Choosing ‘local
database’ will restrict some functionality of the software. Are you sure you
want to continue?”) and “Proceed” in the following “Plug-in” pop-up
window.
4.4.7 Close the newly created database.

4.4.8 Go to the directory in which you placed your database at step 4.4.3 (on
CDC training laptops: C:\Program Files\BioNumerics\Data), open folder for
organism specific assay (for example, “O157MLVA” for STEC O157) and
create two subfolders named “Scripts” and “VNTRtables”.
4.4.9 Save the four MLVA specific scripts (VNTRImport_v4.bns,
VNTRCalc_v4.bns, VNTRReport.bns, VNTRDetails_v2.bns) in the “Scripts”

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

folder and the organism specific assay look-up table (e.g. ABIEcoli.txt) in
the “VNTRtables” folder.
4.4.10 Open the database. The scripts (except the VNTRDetails script) should now
appear under the “Scripts” drop-down menu.
4.5 Importing a peak file for the first time
4.5.1 Review the peak file in Excel: make sure that the ROX labelled molecular
size standard ran properly without skipping any peaks. Remove any data
(failed reactions, controls, internal ladder) that you don’t want to import in
the BioNumerics from the .txt file. Re-name and re-save the modified .txt
file with correct naming format either on your hard drive or on a flash drive.
For example, a modified file of GA140120 would be saved as GA140120-
mod.
4.5.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” drop-
down menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “ABI GeneMapper peak”
file. The script pops up a file dialog box, prompting for the name of the file
to be imported. Select the appropriate file and click “OK”. A second dialog
box pops up, prompting for a variety of other information:
4.5.2.1 Fingerprint file name. This is the name of the fingerprint file that will
be used in BioNumerics. Leave this unchanged, unless a file has
already been imported with the same name. In this case, change the file
name into a new, unique and informative name.
4.5.2.2 Dyes to import. The dye R (Rox) contains only reference markers and
does not need to be imported. Use this list to select what dyes will be
imported; they are B (FAM), G (HEX) and Y (Calred 590). Each dye
will be stored in a different file, resulting from appending the dye
name to the fingerprint file name.
4.5.2.3 Assign reference positions. Leave this option checked.
4.5.2.4 Select imported isolates. Leave this option checked.
4.5.2.5 Pool tags. For this protocol, there are two pool tags representing the
two PCR reactions for each isolate (each loaded in different wells) and
tagged with names “R1” and “R2“. The two tags should appear in the
list. If they do not, they can be added by selecting “add” to update.
4.5.2.6 If everything is filled in appropriately, click “OK” to start the import.
4.5.2.7 NOTE: These settings are automatically saved and reloaded the next
time this script is run.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

4.5.3 When the script is finished, it has:

4.5.3.1 Created a fingerprint type “VNTRFpr” with appropriate settings.
4.5.3.2 Created fingerprint types with appropriate settings for each dye and
each pool used. The names of these fingerprint types are
“VNTRFpr”+”pool name”+”dye” (for example: VNTRFprR1_Y).
4.5.3.3 Created fingerprint files for each dye in the fingerprint type
“VNTRFpr”, and imported the band information in these lanes. The
individual lanes are assigned to their appropriate pool fingerprint type,
according to the known pool tags found in the “Sample File Name”
field.
4.5.3.4 Created new database entries for all isolates found.
4.5.3.5 Linked the fingerprints to their corresponding entries.
4.5.3.6 Selected all isolates for which fingerprint data was imported.
4.6 Determining the copy numbers for the first time (allele assignment)
4.6.1 Run the script VNTRCalc_v4 from the scripts menu. The first time this script
is run, it will automatically create a new character set called “VNTR_vals”.
This character set will hold the copy numbers for each VNTR. The script
will also create a second character set called “VNTR_frags” which holds the
fragment size with the highest fluorescence for each VNTR. Click “OK” on
the two pop up windows notifying that these two character sets will be
created.
4.6.2 Before the script can determine copy numbers from the fragment length
information, it should have sufficient information about what VNTRs can
be found in which pool and dye, what are the repeat lengths, etc. To this
end, the script pops up a dialog box called “VNTR assign”. This dialog box
contains a list of defined VNTRs that is initially empty. To enter the
parameters of the first VNTR, click on “Add”. This brings up a second
dialog box, prompting for all properties of this first VNTR. Refer to

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

Appendices PND16-2a-c for all necessary information to load the

specifications for each organism specificVNTR.

Example: STEC O157

4.6.3 The VNTR information includes:

4.6.3.1 Name. This name will be used for further reference and for the
character name in the character type VNTR_vals. Each VNTR should
have a unique name (for example VNTR_3, VNTR_34, etc).
4.6.3.2 Offset. Each fragment consists of a repeat portion and a constant
portion, due to the fact that the primers do not locate exactly at the start
and the end of the repeat region. This parameter specifies the size (in
base pairs) of the constant portion.
4.6.3.3 Repeat length. This parameter specifies the size of the repeat block unit
(in base pairs).
4.6.3.4 Copy range. These parameters specify the minimum and maximum
number of copies that the script will consider during the copy number
determination.
4.6.3.5 Tolerance. This specifies the maximum difference between the
expected fragment length (calculated by the software) and the actual
length estimated by the sequencer.
4.6.3.6 Take from fingerprint type. This drop-down list should be used to
indicate on what fingerprint type this VNTR was run. The fingerprint
type is determined by the dye and the pool tag.
4.6.4 If all parameters are filled in appropriately, click OK to add the VNTR and
return to the previous dialog box (“VNTR assign”). Repeat the same actions
to enter the information for all VNTRs used for the organism specific
database. When all VNTRs are entered, make sure the box for “Code absent
as negative” is checked. Select appropriate assay specific look-up table (For
example, ABIEcoli” for STEC O157) from the “Fragment ranges” drop-
down menu and press the “Save&Assign” button to let the script assign
copy numbers for the currently selected entries.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

4.6.4.1 NOTE 1: Pressing “Save&Quit” would store the information without

assigning copy numbers for the current selection.
4.6.4.2 NOTE 2: All information regarding the VNTRs is stored with the
database. The next time the script is run, the VNTR definitions will be
loaded automatically.
4.6.4.3 For every fragment with the highest fluorescence level in the
fingerprint type (= the combination of the reaction and the dye), the
script will assign an allele type (copy number) based on the fragment
size ranges specified in the appropriate look-up table (appendices
PND16-1a-c). Note that, within the same fingerprint type, more than
one VNTR can be loaded if there is no overlap within the fragment
size ranges.
4.6.5 When the script has completed, all copy numbers for all VNTRs for the
currently selected entries are determined.
4.6.5.1 NOTE: Two types of problems may arise during the process (if one or
more such errors were encountered during the calculations, an error
report is displayed listing all the problems):
4.6.5.1.1 None of the peaks present in a fingerprint are compatible with
the fragment size ranges in the look-up table. In this case, the
corresponding character value will be scored “-2.0”. In this
situation, run the “VNTRReport” script from the “Scripts” drop-
down menu to verify the reason for the problem:
4.6.5.1.1.1 No amplification (a null allele): mutations, insertions or
deletions in the primer annealing region, or locus located
on a plasmid that was lost. PCR needs to be repeated
only if the null allele occurred in a locus in which null
alleles have not been previously detected (For STEC
O157: VNTR34, VNTR25, VNTR17; and in the case of
VNTR19 for which null alleles are extremely rare, and
hence should always be confirmed. For Salmonella
serotype Typhimurium: ST2, ST3, ST6, ST7 and in the
case of ST8 for which null alleles are extremely rare,
and hence should always be confirmed. For Salmonella
serotype Enteritidis: VNTR5, VNTR8, VNTR9; and in
the case of SE6 for which null alleles are extremely rare
and hence always need to be confirmed).
4.6.5.1.1.2 Fragment size outside the acceptable range (either
slightly outside of range for a previously detected allele
or a possible new allele). Rerun the fragment analysis
reaction to confirm the accuracy and reproducibility of
the sizing. The isolate should be submitted to CDC for
VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

confirmation only if a possible new allele is detected. If

the sizing is reproducibly slightly outside the range for
an existing allele, the CDC database managers will
adjust the look-up table based on the evidence the
submitting laboratory provides.
4.6.5.1.2 More than one peak in a fingerprint is compatible with an
acceptable fragment size range. In this case, the script will use
the solution that corresponds to the peak with the largest “peak
height” value. Possible causes for multiple peaks:
4.6.5.1.2.1 Primer stutter: multiple peaks with sizes 1-2 bp from
each other. No further action required.
4.6.5.1.2.2 Double alleles: two peaks differing by one or more full
repeats from each other. Further actions required for all
other loci except Salmonella serotype Enteritidis locus
SE8 (in a subset of strains double alleles are known to
occur and the major (larger) allele always has the
highest fluorescence):
4.6.5.1.2.2.1 Repeat the PCR with a freshly made template. If
two peaks still observed and the same peak
consistently has the highest fluorescence intensity,
report the predominant peak only. If the same peak
does not consistently have the highest fluorescence
intensity, proceed to the next step.
4.6.5.1.2.2.2 Test ten single colony picks from the culture.
Report the peak that has the highest fluorescence
intensity in the majority of the picks.
4.7 Analyzing a peak file on a routine basis
4.7.1 Review the peak file: make sure that the ROX labelled molecular size
standard ran properly without skipping any peaks. Remove any data (failed
reactions, controls, internal ladder) that you don’t want to import in the
BioNumerics from the txt file. Re-name and re-save the txt file either on
your hard drive or on the flash drive.
4.7.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” drop-
down menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “ABI GeneMapper peak
file”. The script pops up a file dialog box, prompting for the name of the file
to be imported. Select the appropriate file and click “OK”. “VNTR/Import
peak data” dialog box will appear. De-select “R” from the “Dyes to import”
and click “OK”.
4.7.3 Run the script VNTRCalc_v4 from the scripts menu. “VNTR assign” dialog
box will appear. Select appropriate look-up table (For example, “ABIEcoli”
VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

for STECO157) from the “Fragment ranges” drop-down menu and click
“Save&Assign” to assign allele numbers.
4.8 Verifying the allele assignment
4.8.1 For each isolate in the database that has some VNTR data associated, you
can click on the “VNTR frags” and “VNTR vals” entries in the list to open
the “Entry edit” windows.

Example: STEC O157

4.8.2 If the VNTRCalc_v4 script did not detect a fragment for a VNTR, refer to
the step 4.6.5.1.1 of this protocol for details on how to proceed. If the
fragment size is slightly outside the range specified in the look-up table it is
possible to manually assign a temporary allele size and type for that VNTR
until an official confirmation has been performed by CDC.
4.8.2.1 Note: only the CDC database managers are allowed to modify the look-
up table .txt files. Once a modification has been made, the modified
file will be posted on the PulseNet SharePoint site under the QA/QC
manual. An automatic e-mail notification will be sent about the change
in the SOP.
4.8.2.2 To manually assign an allele size and type, click on the fragment size in
the “VNTRfrags” entry edit window or the allele type in the “VNTR
vals” entry edit window. This will open the “Change character value”
window in BioNumerics lower than v5.0. In BioNumerics v5.0 the
allele size or type can be directly highlighted and changed in the entry
edit window (STEC O157 screen shots below).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

4.9 Creating a composite data set to visualize the allele types

4.9.1 In order to display copy numbers next to a dendrogram in a comparison, first
create a “composite data set” that holds the VNTR data.
4.9.1.1 NOTE: The created composite dataset will be automatically saved in the
database, and hence only need to be created once before the first
analysis in the database.
4.9.1.2 From the “Experiments” drop-down menu, select the option “Create new
composite data set…”, enter a name (e.g. VNTR_cmp), and click the
“OK” button. The “Composite data set ‘VNTR_cmp’” window will
appear.
4.9.1.3 Highlight the experiment VNTR_vals and from the “Experiment” drop-
down menu, select the option “Use in composite data set”. Close the
window.
4.9.2 Next time a comparison window is opened (see below step 4.10), there will
be a new experiment VNTR_cmp listed in the bottom of the window
(BioNumerics versions lower than 5.0) or in the top left corner of the
window (BioNumerics version 5.0). This experiment will facilitate the
display of a spreadsheet-like view of the copy numbers (note that it may be
necessary to scroll the experiment list to the right with the arrow button to
bring VNTR_cmp in display in BioNumerics versions lower than 5.0). This
can be shown next to a dendrogram analysis of the data set.
4.10 Performing comparisons based on the VNTR data
4.10.1. The VNTR data contained in the character set VNTR_vals can be analysed
in BioNumerics with all the tools that are available for character data. That
includes cluster analysis with a variety of methods and similarity
coefficients. For VNTR data, the coefficients that make most sense are:
4.10.1.1 Categorical: preferred if differences in copy numbers should be treated
in a qualitative way. This is the only option for creating dendrograms
using MLVA data.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

4.10.1.2 Manhattan: preferred if differences in copy numbers should be treated

in a quantitative way (a larger difference means more distantly related
organisms). This coefficient can be used to construct minimum
spanning trees.
4.10.2 In order to create a dendrogram:
4.10.2.1 Select the isolates to be included in the dendrogram.
4.10.2.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear.
4.10.2.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.2.4 From the “Clustering” drop-down menu, select the option
“Calculate…Cluster analysis (similarity matrix)” and a “Composite
data set comparison” dialog box will appear.
4.10.2.5 Select “Categorical” for “Multi-state coefficient” and “UPGMA” for
“Dendrogram type”.
4.10.2.6 Click on “OK” button to finish the calculations and the “Comparison”
window with the dendrogram will reappear.
4.10.2.7 From the “Layout” drop-down menu, select the option “Show image”.
4.10.2.8 From the “Composite” drop-down menu, select the option ”Show
quantification (values)” and the copy numbers will appear next to the
dendrogram as shown in the STEC O157 screenshot below.

4.10.3 In order to create a minimum spanning tree:

4.10.3.1 Select the isolates to be included in the spanning tree.
4.10.3.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear.
4.10.3.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

4.10.3.4 From the “Clustering” drop-down menu, select the option

“Calculate…Minimum spanning tree (population modeling)” and a
“Minimum spanning tree” dialog box will appear.
4.10.3.5 Make sure the default “Manhattan” is checked for “Coefficient” and
click “OK” and the “Minimum spanning tree” window with the tree
will appear.
4.10.3.6 You can find out the content for each node by clicking on them
individually.

5. FLOW CHART:

6. REFERENCES:
6.1 Hyytia-Trees, E., Lafon, P., Vauterin, P., and Ribot, E. (2010) Multi-laboratory
validation study of standardized multiple-locus VNTR analysis (MLVA)
protocol for Shiga toxin-producing Escherichia coli O157 (STEC O157): a novel
approach to normalize fragment size data between capillary electrophoresis
platforms. Foodborne Path. Dis. 7, 129-136.

7. CONTACTS:
7.1 Eija Trees, D.V.M., Ph.D.
PulseNet Next Generation Subtyping Methods Unit, EDLB, CDC
(404) 639-3672
[email protected]
VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

7.2 Patti Lafon

PulseNet Next Generation Subtyping Methods Unit, EDLB, CDC
(404) 639-2828
[email protected]
7.3 Ashley Sabol
PulseNet Next Generation Subtyping Methods Unit, EDLB, CDC
(404) 639-2947
[email protected]

8. AMENDMENTS:
4/9/2013: appendix PND16-2 (BioNumerics specifications for the STEC O157
VNTR loci) was added.
4/9/2013: Additions to step 4.6.5: more clarification for reasons for error messages
that occur upon allele assignment and guidance for further follow-up for these
errors.
2/26/2014: Merged the SOPs for STEC O157 (PND16), Salmonella serotypes
Typhimurium (PND17) and Enteritidis (PND18) Analysis for Applied Biosystems
Genetic Analyzer 3130/3500 Data into one protocol.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

Appendix PND16-1a

STEC O157 VNTR allele list and corresponding observed fragment size ranges in
the Applied Biosystems Genetic Analyzer 3130/3500
(ABIEcoli look-up table)

Note: This table is posted on the PulseNet SharePoint under the QA/QC manual as a TXT-file so
users may save it locally and use it with the BioNumerics MLVA scripts. Every time the table is
updated, the latest version is posted on SharePoint site.

R1 R2
Count VNTR_3 VNTR_34 VNTR_9 VNTR_25 VNTR_17 VNTR_19 VNTR_36 VNTR_37
1 474-475 122-124
2 339-340 127-128 135-136 283-284
3 343-346 485-486 132-134 140-142 291-292 123-124 157-158
4 349-352 169-171 491-492 138-140 146-148 296-298 129-131 163-165
5 355-358 187-188 495-498 144-146 153-155 302-304 135-137 169-171
6 361-364 205-206 502-504 150-152 159-161 308-310 141-144 175-176
7 367-370 222-224 508-510 156-158 165-167 314-316 150-151 181-183
8 373-376 242-244 514-516 162-164 172-173 320-322 157-158 187-189
9 379-382 260-262 518-522 168-169 177-179 326-328 163-165 192-194
10 385-388 278-280 525-528 174-175 184-185 332-334 170-172 198-200
11 391-394 295-298 530-534 190-191 338-340 177-179 205-206
12 397-401 315-316 536-540 196-197 344-346 184-185 210-212
13 404-406 543-545 191-192 201-203 350-351 191-192 217-218
14 409-412 548-551 356-357 197-199 223-224
15 415-418 554-557 208-209 362-363 204-206 229-231
16 421-426 559-563 211-213 236-237
17 428-430 565-569 218-219 240-244
18 433-435 572-575 225-228 225-226 249-250
19 440-442 578-581 233-234 255-256
20 447-448 584-586 240-242 262-263
21 453-455 590-591 247-248
22 459-461 596-597 273-274
23 466-467 601-603
24 472-473 607-608
25 478-479 613-614

VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

Appendix PND16-1b

S. enterica serotype Typhimurium VNTR allele list and corresponding observed

fragment size ranges in the Applied Biosystems Genetic Analyzer 3130/3500
(ABIST look-up table)

Note: This table is posted on the PulseNet SharePoint under the QA/QC manual as a TXT-file so users
may save it locally and use it with the BioNumerics MLVA scripts. Every time the table is updated, the
latest version is posted on SharePoint site.

R1 R2
Count ST3 ST5 ST7 ST10 ST2 ST6 ST8
0 121-122
1 128-130 205-206
2 177-180 146-147 137-139 322-323
3 149-153 147-151 216-217
4 188-192 160-164 157-158 340-341 222-226
5 200-202 166-168 166-167 345-347 229-231
6 172-174 175-176 351-354 235-236
7 178-179 184-185 358-360 241-243
8 184-185 193-194 364-366 169-170 247-250
9 190-191 202-203 370-372 253-255 298-299
10 195-197 211-212 376-378 259-261
11 201-203 382-384 210-211 265-267 331-332
12 207-208 220-221 271-273
13 213-214 229-230 389-390 277-279
14 219-220 239-240 394-396 256-257 283-285
15 225-226 250-251 401-402 289-290 364-365
16 231-233 258-259 407-409 271-272 295-296 377-378
17 237-239 413-415 286-287 300-302 391-392
18 244-245 420-421 298-299 306-308
19 249-251 286-287 424-427 324-325 312-314 403-405
20 256-257 295-296 431-433 327-334 318-320 424-426
21 261-263 304-305 438-439 351-352 324-326
22 268-269 444-446 362-366 330-332 435-438
23 273-275 321-322 451-453 370-373 336-337 452-458
24 280-281 458-460 377-380 342-343 464-466
25 286-287 464-466 399-401 348-349 471-474
26 292-293 409-410 354-355 491-492

VERSION: REPLACED BY: AUTHORIZED BY:

Page 14 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

27 470-472 360-361 497-499

28 477-478 437-439 366-367 503-504
29 315-316 483-484 448-449 372-373 517-518
30 489-491 469-470 530-532
31 495-496 377-378 537-538
32 501-502 389-390 550-552
33 507-508 556-558
34 514-517 561-565
35 520-521 582-584
36 526-527 589-591
37 609-610
38 615-617
39 622-623
40 545-546

VERSION: REPLACED BY: AUTHORIZED BY:

Page 15 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

Appendix PND16-1c

S. enterica serotype Enteritidis VNTR allele list and corresponding observed fragment
size ranges in the Applied Biosystems Genetic Analyzer 3130/3500
(ABISE look-up table)

Note: This table is posted on the PulseNet SharePoint under the QA/QC manual as a TXT-file so users may
save it locally and use it with the BioNumerics MLVA scripts. Every time the table is updated, the latest
version is posted on SharePoint site.

R1 R2
Count SE1 SE2 SE8 SE6 SE9 SE3 SE5
1 346-350 173-175 160-161
2 163-166 433-436 181-184
3 170-172 300-303 190-192 199-203 172-174
4 177-179 308-309.5 199-201 211-214 177-180
5 184-186 310.4-316.2 224-225 184-185
6 190-193 317.5-324.0 189-191
7 198-199 324.9-331.5 195-197
8 204-206 335-339 408-413 259-260 201-203
9 211-213 342-345 446-447 207-209
10 218-219 348-351 479-482 283-284 213-215
11 225-226 356-358 218-221
12 232-234 363-364 533-534 225-228
13 240-241 370-373 566-567 232-234
14 588-589 238-240
15 255-256 384-385 244-247
16 249-253
17 269-270 258-259
18 264-265
19 268-271
20 274-281
21
22 287-288
23
24 317-319
25
26
27

VERSION: REPLACED BY: AUTHORIZED BY:

Page 16 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

28
29
30 359-360

VERSION: REPLACED BY: AUTHORIZED BY:

Page 17 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

Appendix PND16-2a

BioNumerics specifications for STEC O157 VNTR loci

Reaction R1 R2
Name VNTR_3 VNTR_17
Dye CalRed590 = Y CalRed590 = Y
Offset 321 120
Repeat length 6 6
Copy range 0-25 0-30
Tolerance 2 2
Fragment size in EDL933 379-382 159-161

Name VNTR_34 VNTR_19

Dye FAM = B FAM = B
Offset 100 272
Repeat length 18 6
Copy range 0-20 0-25
Tolerance 3 2
Fragment size in EDL933 278-280 308-310

Name VNTR_9 VNTR_36

Dye FAM = B FAM = B
Offset 465 102
Repeat length 6 7
Copy range 0-50 0-20
Tolerance 2 2
Fragment size in EDL933 530-534 157-158

Name VNTR_25 VNTR_37

Dye HEX = G HEX = G
Offset 110 142
Repeat length 6 6
Copy range 0-20 0-25
Tolerance 2 2
Fragment size in EDL933 138-140 187-189

Fragment size ranges are based on independent runs on multiple instruments at CDC and in PulseNet Participating Laboratories.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 18 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

Appendix PND16-2b

BioNumerics specifications for S. enterica serotype Typhimurium VNTR loci

Reaction R1 R2
Name ST3 ST2
Dye HEX = G FAM = B
Offset 156 62
Repeat length 6* 13**
Copy range 0-20 0-40
Tolerance 2 3
Fragment size in LT2 188-192 370-373
Copy number in LT2 4* 23**

Name ST5 ST6

Dye FAM = B CalRed590 = Y
Offset 139 191
Repeat length 6 6
Copy range 0-60 0-50
Tolerance 2 2
Fragment size in LT2 219-220 271-273
Copy number in LT2 14 12

Name ST7 ST8

Dye CalRed590 = Y HEX = G
Offset 115 196
Repeat length 9 11***
Copy range 0-30 0-50
Tolerance 3 3
Fragment size in LT2 157-158 556-558
Copy number in LT2 4 33***

Name ST10
Dye HEX = G
Offset 311
Repeat length 6
Copy range 0-50
Tolerance 2
Fragment size in LT2 376-378
Copy number in LT2 10
* A half of a repeat difference considered significant. Actual repeat length 12 bp and actual copy number in LT2 2.5
** A third of a repeat difference considered significant. Actual repeat length 39 bp and actual copy number in LT2 8.0
*** A third of a repeat difference considered significant. Actual repeat length 33 bp and actual copy number in LT2 11.0

Fragment size ranges are based on independent runs on multiple instruments at CDC and in PulseNet Participating Laboratories.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 19 of 20
 PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF CODE:
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157 PND16
(STEC O157) AND SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM
Effective Date:
AND ENTERIDITIS IN BIONUMERICS-APPLIED BIOSYSTEMS GENETIC 02 26 14
ANALYZER 3130/3500 DATA

Appendix PND16-2c

BioNumerics specifications for S. enterica serotype Enteritidis VNTR loci

Reaction R1 R2
Name SE1 SE9
Dye B (FAM) B (FAM)
Offset 149 165
Repeat length 7 9
Copy range 0-10 0-5
Tolerance 2 2
Fragment size in K1891 190-193 181-184
Copy number in K1891 6 2

Name SE2 SE3

Dye Y (Calred 590) Y (Calred 590)
Offset 280 165
Repeat length 7 12
Copy range 0-20 0-10
Tolerance 2 3
Fragment size in K1891 335-339 211-215
Copy number in K1891 8 4

Name SE8 SE5

Dye G (HEX) G (HEX)
Offset 261 154
Repeat length 86 6
Copy range 0-3 0-25
Tolerance 4 2
Fragment size in K1891 433-436 201-203
Copy number in K1891 2 8

Locus SE6
Dye B (FAM)
Offset 149
Repeat length 33
Copy range 2-20
Tolerance 3
Fragment size in K1891 479-482
Copy number in K1891 10

Fragment size ranges are based on independent runs on multiple instruments at CDC and in PulseNet Participating Laboratories.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 20 of 20
READING CONTROL SHEET FOR:
STANDARD OPERATING PROCEDURES FOR THE PULSENET DATABASES
(PND)

NAME DATE COMMENTS SIGNATURE

By signing above, you are indicating that you have read and understood all SOPs
included in the PND section of this manual.
 CODE: PNQ01
STANDARD OPERATING PROCEDURE FOR TIFF QUALITY GRADING Effective Date:
5 09 2005

1. PURPOSE: To describe guidelines for the quality of TIFF images submitted to the PulseNet national
databases.

2. SCOPE: This applies to all TIFF images submitted to PulseNet, thereby allowing comparison of results
with other PulseNet laboratories.

3. DEFINITIONS/TERMS:

3.1 TIFF: Tagged Image File Format

3.2 TIFF Quality: The grading of the appearance and ease of analysis of a TIFF, according to the TIFF
Quality Grading Guidelines within this SOP. This is a main component of the evaluation of a TIFF
submitted for certification or proficiency testing.
3.3 SOP: Standard Operating Procedure

4. RESPONSIBILITIES/PROCEDURE:

TIFF Quality Grading Guidelines

Parameter
Excellent Good Fair Poor
Image By protocol, for - Gel doesn’t fill Not protocol; for example, Not protocol; for example, >1
Acquisition example: whole TIFF but band one of the following: of the following:
and Running - Gel fills whole finding is not affected - Gel doesn’t fill whole TIFF - Gel doesn’t fill whole TIFF
Conditions TIFF and band finding is affected and this affects band finding
- Wells included on - Wells not included on TIFF - Wells not included on TIFF
TIFF - Bottom band of standard not - Bottom band of standard not
- Bottom band of 1-1.5 cm from bottom of gel 1-1.5 cm from bottom of gel
standard 1-1.5 cm - Band spacing of standards - Band spacing of standards
from bottom of gel doesn’t match global standard doesn’t match global standard
Cell The cell 1-2 lanes contain - >2 lanes contain darker or The cell concentrations are
Suspensions concentration is darker or lighter lighter bands than the other uneven from lane to lane,
approximately the bands than the other lanes, or making the gel impossible to
same in each lane lanes - At least 1 lane is much analyze
darker or lighter than the
other lanes, making the gel
difficult to analyze
Bands Clear and distinct - Slight band - Some band distortion (e.g., - Band distortion that makes
all the way to the distortion in 1 lane nicks) in 2-3 lanes but still analysis difficult
bottom of the gel but doesn’t interfere analyzable - Very fuzzy bands.
with analysis - Fuzzy bands - Many bands too thick to
- Bands are slightly - Some bands (e.g., 4-5) are distinguish
fuzzy and/or slanted too thick - Bands at the bottom of the
- A few bands (e.g., - Bands at the bottom of the gel too light to distinguish
≤3) difficult to see gel are light, but analyzable
clearly (e.g., DNA
overload), especially
at bottom of gel
Lanes Straight - Slight smiling - Significant smiling - Smiling or curving that
(higher bands in the - Slight curves on the outside interferes with analysis
outside lanes vs. the lanes
inside) - Still analyzable
- Lanes gradually run
longer toward the
right or left
- Still analyzable

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 2
 CODE: PNQ01
STANDARD OPERATING PROCEDURE FOR TIFF QUALITY GRADING Effective Date:
5 09 2005

Restriction Complete - One to two faint - One lane with many shadow - Greater than 1 lane with
restriction in all shadow bands on gel bands several shadow bands
lanes - A few shadow bands spread - Lots of shadow bands over
out over several lanes the whole gel
Gel Clear - Mostly clear - Some debris present that - Lots of debris present that
Background background may or may not make may or may not make
- Minor debris analysis difficult (e.g., auto analysis difficult (i.e., auto
present that doesn’t band search finds too many band search finds too many
affect analysis bands) bands)
- Background caused by
photographing a gel with very
light bands (image contrast
was “brought up” in
photographing gel-makes
image look grainy)

DNA Not present - Minor background - Significant smearing in 1-2 - Significant smearing in >2
Degradation (smearing) in a few lanes that may or may not lanes that may or may not
(smearing in lanes but bands are make analysis difficult make analysis difficult
the lanes) clear - Minor background - Smearing so that a lane is
(smearing) in many lanes not analyzable (except if
untypeable [thiourea
required])

5. FLOW CHART:

6. BIBLIOGRAPHY:

7. CONTACTS:

8. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 2
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

1. PURPOSE: To describe the procedure for certifying PulseNet personnel to enable full
participation in PulseNet activities, including on-line access to the PulseNet National
Databases.

2. SCOPE: This procedure applies to all PulseNet personnel performing PFGE, image
acquisition, and/or BioNumerics gel analysis.

3. DEFINITIONS/TERMS:

3.1. PFGE: Pulsed-field Gel Electrophoresis

3.2. CDC: Centers for Disease Control and Prevention
3.3. QA/QC: Quality Assurance/Quality Control
3.4. SOP: Standard Operating Procedure
3.5. BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths,
Belgium
3.6. TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in
BioNumerics
3.7. Bundle file: A file with a .bdl extension that is produced in BioNumerics and contains the
analysis of at least one lane of a gel image
3.8. Gel-certified or TIFF-certified: An individual or laboratory that is certified in laboratory
methods for PFGE and image acquisition
3.9. Analysis-certified: An individual who is certified in BioNumerics gel analysis
3.10. Certification files: TIFF and/or bundle files submitted by PulseNet participants for
certification evaluation.
3.11. Certification file evaluator: An individual who evaluates certification files
3.12. PulseNet Area Laboratory: Laboratory, designated by CDC, which has agreed to assume
responsibility for additional PulseNet duties for laboratories within their support zone. The
current Area Laboratories include MA, MN, WA, TX, VA, UT, MI, and CDC.

4. RESPONSIBILITIES:

4.1 Individuals performing PulseNet-related work (i.e., preparing PFGE gels and/or analyzing TIFFs of
PFGE gels) must submit certification file(s) and have them reviewed before being able to submit
TIFF images and BioNumerics analyses to the PulseNet National Databases.
4.1.1 Submitted certification files must document the submitter’s highest level of competence in
producing and imaging PFGE gels and/or in analyzing TIFFs of PFGE gels.
4.1.2 Individuals can be certified for each PulseNet organism in one of three ways:
4.1.2.1 Gel only (i.e., gel-certified). An individual can submit TIFFs to an analysis-certified person
for subsequent analysis and uploading to the national databases.
4.1.2.2 Analysis only (i.e., analysis-certified). An individual can analyze TIFFs of gels generated
by TIFF-certified individuals and upload those analyzed images to the national databases.
4.1.2.3 Both gel and analysis (i.e., gel and analysis-certified). An individual can perform PFGE,
image acquisition, and BioNumerics analysis of TIFFs, uploading the analyses to the
national databases.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

4.1.3 For each PulseNet organism, at least one person from each PulseNet participating laboratory
should be gel-certified and one person should be analysis-certified. One person can be both gel
and analysis-certified. Laboratories cannot have analysis-certified personnel without gel-certified
personnel; gel certification must occur before or concurrent with analysis certification.
4.2 Individuals performing PulseNet-related work at CDC must submit certification file(s) and have
them reviewed before being able to submit TIFF images and BioNumerics analyses to the PulseNet
National Databases.
4.2.1 All PulseNet CDC patterns must be uploaded directly from a client database that is housed on the
PulseNet network drive. Those at CDC without access to a client database on the PulseNet
network drive may not upload PFGE patterns.

5. PROCEDURE:

5.1 PulseNet participants request certification set(s) from CDC (via [email protected]) if they do not already
have them.
5.1.1 Currently, certification sets are available for E. coli O157:H7, E. coli Non O157 STEC (analysis-
only), Salmonella, Shigella, Listeria monocytogenes, Campylobacter jejuni, Vibrio cholerae,
Vibrio parahaemolyticus and C.botulinum.
5.2 CDC sends the requested certification set(s) and detailed instructions for the gel preparation and
analysis of the certification set organisms. See Appendices PNQ02-1 through PNQ02-9.
5.3 Individuals in each laboratory who wish to be gel-certified make plugs for each sample and run gel(s)
with the proper controls.
5.4 Individuals in the laboratory who wish to be analysis-certified analyze TIFF(s) produced using the
certification set instructions and create a bundle file of their analyses, according to the certification set
instructions.
5.5 TIFF file(s) are submitted to CDC for review. See Appendices PNQ02-1 through PNQ02-9 for
submission instructions.
5.6 Bundle files are submitted to CDC for review. See Appendices PNQ02-1 through PNQ02-9 for
submission instructions.
5.7 Submitters are notified in writing of the results of their certification file(s) evaluation. (See PNQ03
for more information on the evaluation of PulseNet certification files.)
5.7.1 If the submitted certification files pass the certification evaluation, the submitter is considered
certified as long as they remain in their current laboratory and that laboratory successfully
completes annual proficiency testing (See PNQ04 for more information on the PulseNet
proficiency testing program). If a person relocates to a different PulseNet laboratory, they must be
recertified.
5.7.2 If the submitted certification files do not pass the certification evaluation
5.7.2.1 The individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved
5.7.2.2 At any point, if the evaluator and/or CDC feels that the individual needs additional
troubleshooting and/or training before resubmission, the Area Laboratory responsible for
the individual’s region will be notified
5.7.2.3 If the submitter fails certification three times, the individual will not be allowed to submit
again for six months. Before resubmitting, the individual will be expected to work with
CDC and/or their PulseNet Area Laboratory until satisfactory results are achieved. This
includes, but is not limited to troubleshooting and training in the PulseNet PFGE protocols,
BioNumerics and the PulseNet masterscripts.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:
8.1 PulseNet Database Unit Chief: Kelley Hise, MPH
(404) 639-0704
[email protected]
8.2 CDC PulseNet Database Administration Team
(404) 639-4558
[email protected]

9. AMENDMENTS:
9.1 Appendix PNQ02-6 added on 5/2/2007
9.2 Appendix PNQ02-7 added on 11/18/2008
9.3 Appendix PNQ02-8 added on 10/6/2009
9.4 As of 10/6/2009 the Shigella certification set (Appendix PNQ02-3) no longer includes isolates 92-01,
93-01 or 96-01.
9.5 As of 10/6/2009 the Campylobacter jejuni certification set (Appendix PNQ02-5) no longer includes
isolates D996 or D2253.
9.6 1/5/2010 Appendix PNQ02-6 section 4 was updated to correct lane information for the test strains
9.7 Appendix PNQ02-9 for C. botulinum certification added on 7/15/2010
9.8 July 2010 added « Images that have a file size that is not within the range of ~300-400 kb will be
deemed unsatisfactory and certification file evaluators will ask for a resubmission.” to all appendices.
9.9 July 2010 added section 4.2.
9.10 November 2010 typos for H9812 were corrected in all the lane assignment tables.
9.11 Appendix PNQ02-8 was updated on 3/4/2013 to reflect changes to the normalization step during
analysis of Non O157 images. Please mark standard lanes as shown in the reference lane (top of 668.9
Kb and the top band of 167.1 Kb).
9.12 Appendix PNQ02-10 was added for Shigella flexneri analysis certification instructions

Appendix PNQ02-1

Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) E. coli O157:H7 Cultures

Biological Safety Warning: E. coli O157:H7 strains are considered Level 2 biological agents by the U.S.
Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out
laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.

Note: Store the lyophilized cultures at 4oC in the dark until they are reconstituted.

Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop

Reagents Needed:
VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media

Sterile grade reagent water
70% isopropyl alcohol

Procedure for Reviving Cultures:

Day 1

1. Document the isolate number(s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.

2. Re-suspend the lyophilized cells with 1ml of sterile grade reagent water. Allow to stand for a few minutes and/or
mix gently to produce a uniform suspension. With an inoculating loop, streak a small amount of this suspension onto
a blood agar plate (BAP) and incubate at 37ºC overnight.

Days 2 and 3

Check the BAP; if the culture appears pure, pick an isolated colony, and inoculate a fresh BAP for heavy growth;
incubate at 37oC overnight. Use the growth from this plate to make PFGE plugs of the standard strains. Before
making the plugs, transfer culture to fresh medium and incubate at 37oC overnight; this will ensure that the same
culture can be retested, if necessary.

After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let me know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze
(-70°C) or stock these strains according to your laboratory’s policy within one week of receiving them. Then
your laboratory will have stock cultures of this PulseNet certification set for future use, including the
PulseNet certification of additional personnel.

The strain numbers of the E. coli cultures are as follows:

CDC16-98 CDC20-98 CDC24-98 CDC68-98

Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Escherichia coli,
non-typhoidal Salmonella serotypes and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE)”. Detailed
instructions for making the PFGE plugs can be found in the PulseNet QA/QC Manual PNL05. This document is
available in the QA/QC conference on CDC Team or at www.cdc.gov/pulsenet/. If you can not access the protocol,
please request it by sending an E-mail to [email protected].

1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.
2. Restrict one plug slice from the four test cultures (CDC16-98, CDC20-98, CDC24-98, and
CDC68-98) and 3 plug slices of Salmonella ser. Braenderup H9812 (the PulseNet Universal
Standard Strain) with XbaI (40-50 Units/plug slice) for 2 hours at 37°C..
3. Restrict one plug slice from three of the test cultures (CDC16-98, CDC20-98, and CDC24-98)
with AvrII (BlnI; 25-30 Units/plug slice) for 2 hours at 37°C.
4. Load H9812 in Lanes 1, 5, 10 and the test strains as follows:
VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

Lane Certification Strain Enzyme

1 H9812 XbaI
2 16-98 XbaI
3 20-98 XbaI
4 24-98 XbaI
5 H9812 XbaI
6 16-98 BlnI
7 20-98 BlnI
8 24-98 BlnI
9 68-98 XbaI
10 H9812 XbaI

Note: If you use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty.
Although the use of the ~0.5-mm wide 15-teeth combs [Bio-Rad, 170-4326] in the standard casting stand
[14 x 13-cm] is not recommended for routine PFGE analysis of test isolates, the use of the smaller comb
teeth will be allowed for certification.

5. Run the gel using the E. coli electrophoresis conditions. These run times are based on the
equipment and reagents used at CDC. If the gels generated in your lab do not have the lowest
band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run time may have to
be changed.

6. Staining, de-staining, and gel documentation (imaging):

a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns that will automatically
be asked to rerun the gel and submit a new certification TIFF. Images that have a file size that
is not within the range of ~300-400 kb will be deemed unsatisfactory and certification file
evaluators will ask for a resubmission.

b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persists repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme.

7. Bundle file creation

a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database
entries as follows: 3 test strains restricted with XbaI and BlnI and one test strain restricted
only with XbaI. If there are more than 4 entries in the bundle file, one or more lanes have not
been linked properly.

b. Create the bundle file using the lightning bolt icon.(refer to the Gel Analysis Guidelines
PND04 for instructions on how to create a PulseNet Bundle File). Bundles created using this
icon are “PulseNet bundles” and will contain only standardized PulseNet experiments and
fields. “PulseNet bundles” can be recognized by the “PN” that is automatically added to the
file name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

8. Send the TIFF file and/or bundle (*.bdl) file of your gel images to CDC Pulse Net at [email protected]
within four weeks after receiving the strains.

a. In the email to CDC, include E. coli Certification Gel in the Subject line, so it can be forwarded to
the correct person. Include the isolate number and restriction enzyme used for each lane on the gel
in the body of the email. If you are sending certification TIFFs and bundle files for more than one
organism, please send one email per organism.

b. Name the TIFF and bundle files of your gel images as follows:

TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to
four letters of the file. The next 2 spaces will indicate the year the file was created. The
next 3 spaces indicate the sequential number of the file submitted from your laboratory
during a calendar year. For example: GA09012.tif is the twelfth file submitted from the
state of Georgia during 2009.

Bundle: The bundle files are named in a similar manner as the TIFF files with the first two
to four letters of the file name indicating the unique identifier code for your laboratory. The
next 2 spaces indicate the year the bundle file was created, and the next 3 spaces indicate
the sequential bundle file number from each laboratory. For example, the eighth bundle file
submitted from the state of Georgia during 2009 would be named GA09008PN.bdl.

For each PulseNet pathogen, an individual may be certified for:

a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.

After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.

Appendix PNQ02-2

Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Salmonella Cultures

Biological Safety Warning: Salmonella strains are considered Level 2 biological agents by the U.S. Department of
Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out laboratory
work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.

Note: Store the lyophilized cultures at 4oC in the dark until they are reconstituted.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop

Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water
70% isopropyl alcohol

Procedure for Reviving Cultures:

Day 1

1. Document the isolate number(s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.

2. Re-suspend the lyophilized cells with 1ml of sterile grade reagent water. Allow to stand for a few minutes and/or
mix gently to produce a uniform suspension. With an inoculating loop, streak a small amount of this suspension onto
a blood agar plate (BAP) and incubate at 37ºC overnight.

Days 2 and 3

1. Check the BAP; if the culture appears pure, pick an isolated colony, and inoculate a fresh BAP for heavy growth;
incubate at 37oC overnight. Use the growth from this plate to make PFGE plugs of the standard strains. Before
making the plugs, transfer culture to fresh medium and incubate at 37oC overnight; this will ensure that the same
culture can be retested, if necessary.

After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let me know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze
(-70°C) or stock these strains according to your laboratory’s policy within 1 week of receiving them. Then,
your laboratory will have stock cultures of this PulseNet certification set for future use, including the
PulseNet certification of additional personnel.

The strain numbers of the Salmonella cultures are as follows:

CDC61-99 CDC78-99 CDC87-03 CDC98-03

Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Escherichia coli,
non-typhoidal Salmonella serotypes and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE).” Detailed
instructions for making the PFGE plugs can be found in the PulseNet QA/QC Manual PNL05. This document is
available in the QA/QC conference on CDC Team or at www.cdc.gov/pulsenet/. If you can not access the protocol,
please request it by sending an E-mail to [email protected].

1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

2. Restrict one plug slice from the four test cultures (CDC61-99, CDC78-99, CDC87-03, and
CDC98-03) and 3 plug slices of Salmonella ser. Braenderup H9812 (the PulseNet Universal
Standard Strain) with XbaI (40-50 Units/plug slice) for 2 hours at 37°C.
3. Restrict one plug slice from three of the test cultures (CDC61-99, CDC78-99, and CDC87-03)
with AvrII (BlnI; 25-30 Units/plug slice) for 2 hours at 37°C.
4. Load H9812 in Lanes 1, 5, 10 and the test strains as follows:

Lane Certification Strain Enzyme

1 H9812 XbaI
2 61-99 XbaI
3 78-99 XbaI
4 87-03 XbaI
5 H9812 XbaI
6 61-99 BlnI
7 78-99 BlnI
8 87-03 BlnI
9 98-03 XbaI
10 H9812 XbaI

Note: If you use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty.
(The use of the ~0.5-mm wide 15-teeth combs [Bio-Rad, 170-4326] in the standard casting stand [14 x 13-
cm] is not recommended for routine PFGE analysis of test isolates, but will be allowed for certification).

5. Run the gel using the Salmonella electrophoresis conditions. The run times listed in the protocol
are based on the equipment and reagents used at CDC. If the gels generated in your lab do not
have the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run
time may have to be changed.

6. Staining, de-staining, and gel documentation (imaging):

a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting a certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns will automatically be
asked to rerun the gel and submit a new certification TIFF. Images that have a file size that is
not within the range of ~300-400 kb will be deemed unsatisfactory and certification file
evaluators will ask for a resubmission.

b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persists repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme.

7. Bundle file creation

a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database
entries as follows: 3 test strains restricted with XbaI and BlnI and one test strains restricted
only with XbaI. If there are more than 4 entries in the bundle file, one or more lanes have not
been linked properly.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

b. Create the bundle file using the lightning bolt icon.(refer to the Gel Analysis Guidelines
PND04 for instructions on how to create a PulseNet Bundle File). Bundles created using this
icon are “PulseNet bundles” and will contain only standardized PulseNet experiments and
fields. “PulseNet bundles” can be recognized by the “PN” that is automatically added to the
file name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.

8. Send the TIFF file and/or bundle (*.bdl) file of your gel images to CDC Pulse Net at
[email protected] within four weeks after receiving the strains.

a. In the email to CDC, include Salmonella Certification Gel in the Subject line, so it can be
forwarded to the correct person. Include the isolate number and restriction enzyme used for
each lane on the gel in the body of the email. If you are sending certification TIFFs and
bundle files for more than one organism, please send one email per organism.

b. Name the TIFF and bundle files of your gel images as follows:

TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to
four letters of the file. The next 2 spaces will indicate the year the file was created. The next
3 spaces indicate the sequential number of the file submitted from your laboratory during a
calendar year. For example: GA09012.tif is the twelfth file submitted from the state of
Georgia during 2009.

Bundle: The bundle files are named in a similar manner as the TIFF files with the first two
to four letters of the file name indicating the unique identifier code for your laboratory. The
next 2 spaces indicate the year the bundle file was created, and the next 3 spaces indicate
the sequential bundle file number from each laboratory. For example, the eighth bundle file
submitted from the state of Georgia during 2009 would be named GA09008PN.bdl.

For each PulseNet pathogen, an individual may be certified for:

a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.

After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts

Appendix PNQ02-3

Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Shigella Cultures

VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

Biological Safety Warning: Shigella strains are considered Level 2 biological agents by the U.S. Department of
Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out laboratory
work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.

Note: Store the lyophilized cultures at 4oC in the dark until they are reconstituted.

Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop

Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water
70% isopropyl alcohol

Procedure for Reviving Cultures:

Day 1

1. Document the isolate number(s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.

2. Re-suspend the lyophilized cells with 1ml of sterile grade reagent water. Allow to stand for a few minutes and/or
mix gently to produce a uniform suspension. With an inoculating loop, streak a small amount of this suspension onto
a blood agar plate (BAP) and incubate at 37ºC overnight.

Days 2 and 3

Check the BAP; if the culture appears pure, pick an isolated colony, and inoculate a fresh BAP for heavy growth;
incubate at 37oC overnight. Use the growth from this plate to make PFGE plugs of the standard strains. Before
making the plugs, transfer culture to fresh medium and incubate at 37oC overnight; this will ensure that the same
culture can be retested, if necessary.

After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let me know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze
(-70°C) or stock these strains according to your laboratory’s policy within 1 week after receiving them. Then,
your laboratory will have stock cultures of this PulseNet certification set for future use, including PulseNet
certification of additional personnel.

Biosafety Warning: Shigella species have a low infectious dose and are demonstrated hazards to laboratory
personnel; please use extreme caution and Biosafety Level 2 practices (at a minimum) when transferring and
handling Shigella strains. Work in a biological safety cabinet when handling large amounts of cells. Disinfect or
dispose of all plasticware and glassware that comes in contact with the cultures in a safe and appropriate manner.

The strain numbers of the Shigella sonnei cultures are as follows:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

CDC90-01 CDC91-01 CDC94-01 CDC95-01

Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Escherichia coli,
non-typhoidal Salmonella serotypes and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE)” Detailed
instructions for making the PFGE plugs can be found in the PulseNet QA/QC Manual PNL06. This document is
available in the QA/QC conference of the CDC Team Support or at www.cdc.gov/pulsenet/. If you can not access
the protocol, please request it by sending an E-mail to [email protected].

1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.
2. Restrict one plug slice from each of the 4 test cultures (CDC90-01, CDC91-01, CDC94-01, and
CDC95-01) and 3 plug slices of H9812 (the PulseNet Universal Standard Strain) with XbaI (40-50
Units per plug slice) for 2 hours at 37ºC.
3. Restrict one plug slice from three of the test cultures (CDC90-01, CDC91-01, and CDC94-01)
with AvrII (BlnI; 25-30 Units/plug slice) for 2 hours at 37°C.
4. Load H9812 in Lanes 1, 6, 10 and the test strains as follows:

Lane Certification Strain Enzyme

1 H9812 XbaI
2 CDC90-01 XbaI
3 CDC91-01 XbaI
4 CDC94-01 XbaI
5 CDC95-01 XbaI
6 H9812 XbaI
7 CDC90-01 BlnI
8 CDC91-01 BlnI
9 CDC94-01 BlnI
10 H9812 XbaI

Note: If you use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty
(Although the use of the ~0.5-mm wide 15-teeth combs [Bio-Rad, 170-4326] in the standard casting stand
[14 x 13-cm] is not recommended for routine PFGE analysis of test isolates, the use of the smaller comb
teeth will be allowed for certification.).

5. Run the gel using the Shigella electrophoresis conditions. The run times listed in the protocol are
based on the equipment and reagents used at CDC. If the gels generated in your lab do not have
the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run time
may have to be changed.

6. Staining, de-staining, and gel documentation (imaging):

a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns that will automatically
be asked to rerun the gel and submit a new certification TIFF. Images that have a file size that
is not within the range of ~300-400 kb will be deemed unsatisfactory and certification file
evaluators will ask for a resubmission.

b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persists, repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme.

7. Bundle file creation

a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database
entries as follows: 3 test strains restricted with XbaI and BlnI and one test strain restricted
only with XbaI. If there are more than 4 entries in the bundle file, one or more lanes have not
been linked properly.

b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines PND04
for instructions on how to create a PulseNet Bundle File). Bundles created using this icon are
“PulseNet bundles” and will contain only standardized PulseNet experiments and fields.
“PulseNet bundles” can be recognized by the “PN” that is automatically added to the file
name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.

8. Send the TIFF file and/or bundle (*.bdl) file of your gel images to CDC PulseNet at [email protected].

a. In the e-mail to CDC, include Shigella Certification in the subject line, so it can be forwarded
to the correct person. Include the isolate number and restriction enzyme used for each lane on
the gel in the body of the email. If you are sending certification TIFFs and bundle files for
more than one organism, please send one email per organism.

b. Name the TIFF and bundle files of your gel images as follows:

TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to four
letters of the file. The next 2 spaces will indicate the year the file was created. The next 3 spaces
indicate the sequential number of the file submitted from your laboratory during a calendar year.
For example: GA09012.tif is the twelfth file submitted from the state of Georgia during 2009.

Bundle: The bundle files are named in a similar manner as the TIFF files with the first two to four
letters of the file name indicating the unique identifier code for your laboratory. The next 2 spaces
indicate the year the bundle file was created, and the next 3 spaces indicate the sequential bundle
file number from each laboratory. For example, the eighth bundle file submitted from the state of
Georgia during 2009 would be named GA09008PN.bdl.

For each PulseNet pathogen, an individual may be certified for:

a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.

After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.

Appendix PNQ02-4
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Listeria monocytogenes
Cultures

Biological Safety Warning: Listeria monocytogenes is considered a Level 2 biological agent by the U.S.
Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry
out laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.

Note: Store the lyophilized culture at 4˚C in the dark until they are reconstituted.

Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 μl sterile inoculating loop

Reagents Needed:
Blood agar plate or equivalent media
Brain heart infusion agar plates
Sterile grade reagent water
70% isopropyl alcohol

Procedure for Reviving Cultures:

1. Wipe the aluminum cover and outside of the vial with isopropyl alcohol. Using forceps, aseptically remove
the aluminum cover and rubber stopper from the vial containing the lyophilized culture. Wipe the outside
of the vial and stopper with alcohol after the metal cap is removed.

2. Re-suspend the lyophilized cells with 1.0 mL of sterile reagent grade water. Allow to stand for a few
minutes and/or mix gently to produce a uniform suspension. Pipet 10µl of Listeria cell suspension onto a
blood agar plate (BAP) and with a 10µl loop streak for growth. Incubate at 37ºC overnight.

3. Check the blood agar plate; if the culture appears pure, pick 2-3 representative colonies and inoculate a
fresh brain heart infusion agar plate for heavy growth; incubate at 37˚C for 18 to 24 hours. Use the growth
from the brain heart infusion agar plate to make agarose gel plugs as described in the Standardized
Molecular Subtyping of Foodborne Bacterial Pathogens by Pulsed-Field Gel Electrophoresis.

Listeria monocytogenes certification isolate plug slice loading position:

Lane number Isolate number Restriction Enzyme

1 S. Braenderup H9812 XbaI

VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

2 CDC-02-H8393 AscI
3 CDC-03-H8394 AscI
4 CDC-04-H8395 AscI
5 Lm control H2446 AscI
6 S. Braenderup H9812 XbaI
7 CDC-02-H8393 ApaI
8 CDC-03-H8394 ApaI
9 CDC-04-H8395 ApaI
10 S. Braenderup H9812 XbaI

Recommended gel casting bed and comb: Standard casting stand, 14 x 13 cm frame/platform and 10 well comb, 14
cm wide, 1.5-mm thick.
After the strains have been reconstituted according to the above instructions, streak each culture to a Trypticase Soy
+ 5% Sheep’s blood agar plate (BAP) and allow them to incubate at 37°C for 24 hours to check for viability. Please
let me know if the shipment does not arrive in satisfactory condition, or if the cultures are not viable. Freeze (-70˚C)
or stock these strains immediately after reviving them according to your laboratory policy. The L. monocytogenes
strains in the revised set are:

CDC#H8393 CDC#H8394 CDC#H8395 CDC#H2446 control strain

Please follow the Listeria monocytogenes standardized protocol (PNL04) for making the plugs and running the gel
for these isolates. Refer to the Standardized Laboratory Protocol for Molecular Subtyping of Foodborne Bacterial
Pathogens by PFGE (Section 5.3, Revised April, 2002).

Salmonella ser. Braenderup strain (H9812) is used as the reference standard. DNA of the H9812 strain must be
digested with XbaI to give the appropriate band pattern. Follow instructions in the E. coli O157:H7 Standardized
Laboratory protocol (PNL05) for making S. Braenderup plugs. If you do not have the S. Braenderup reference
standard you may request it from [email protected]. The chart shown above lists the positions where plug slices
should be loaded on the gel.

1. Staining, de-staining, and gel documentation (imaging):

a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns that will automatically
be asked to rerun the gel and submit a new certification TIFF. Images that have a file size that
is not within the range of ~300-400 kb will be deemed unsatisfactory and certification file
evaluators will ask for a resubmission.

b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persists, repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme.

Follow the instructions in steps 1-4 below for analysis, creating the bundle (*.bdl) file, and sending the TIFF(s)
and/or bundle file of your Listeria monocytogenes gel image(s) to CDC for certification.

1.In the bundle file, do not include lanes from more than two gels. Enter the corresponding CDC strain
number in the BioNumerics “Key” field. The bundle file should contain 4 database entries as follows:
VERSION: REPLACED BY: AUTHORIZED BY:

Page 14 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

3 test strains restricted with AscI and ApaI and 1 test strain restricted only with AscI. If there are more
than 4 entries in the bundle file, one or more lanes have not been linked properly.

2. Create the bundle file using the lightning bolt icon (see page 104 in the Appendix of the Dec. 2003
CDC BioNumerics manual). Bundles created using this icon are “PulseNet bundles” and will contain
only standardized PulseNet experiments and fields. “PulseNet bundles” can be recognized by the
“PN” that is automatically added to the file name. If a non-PulseNet bundle is submitted, you will
automatically be asked to resubmit.

3. Send the TIFF file and/or bundle file of your gel images to CDC PulseNet at [email protected].

a. In the e-mail to CDC, include the appropriate description of the gel (Listeria Certification) in
the subject line, so it can be forwarded to the correct person. Include the isolate number and
restriction enzyme used for each lane on the gel in the body of the e-mail or as an attachment.
If you are sending certification TIFFs and bundle files for more than one organism, please
send one e-mail per organism.

b. Name the TIFF and bundle files of your gel images as follows:

TIFF: Use he unique identifier code that was assigned by CDC PulseNet for the first two,
three, or four letters of the file. The next 2 spaces will indicate the year the file was submitted.
The next 3 spaces indicate the sequential number of the file submitted from your laboratory
during a calendar year. For example: GA09012.tif is the twelfth file submitted from the state
of Georgia during 2009.

Bundle: The bundle files are named in a similar manner as the TIFF files with the first two,
three, or four letters of the file name indicating the unique identifier code for your laboratory.
The next 2 spaces indicate the year the bundle file was submitted, and the next 3 spaces
indicate the sequential bundle file number from each laboratory. For example, the eighth
bundle file submitted from the state of Georgia during 2009 would be named
GA09008PN.bdl.

c. Please refer to Section 15 of the PulseNet PFGE Manual for additional information on naming
the TIFF and bundle files and submitting gel images to PulseNet.

4. For each PulseNet pathogen, an individual may be certified for:

a. TIFF image only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database)
c. Both TIFF and analysis

After the gel images are submitted, the certification file evaluator will analyze the gels and inform your laboratory of
your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the TIFF images
are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for analysis; if
the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID device (also
called a fob) and will be granted on-line access to the appropriate PulseNet national database. (Up to two SecurID
devices can be provided to a participating public health PulseNet laboratory. SecurID devices are issued to
individuals within a laboratory. The devices cannot be shared and must be returned to CDC if the certified individual
leaves his or her position in the laboratory.) If the submitted certification files are not satisfactory, the individual will
need to review the troubleshooting comments received from the evaluator and resubmit once results have improved.
If the submitter fails certification three times, the individual will not be allowed to submit again for six months.
Before resubmitting, the individual will be expected to work with CDC and/or their PulseNet Area Laboratory until

VERSION: REPLACED BY: AUTHORIZED BY:

Page 15 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

satisfactory results are achieved. This includes, but is not limited to troubleshooting and training in the PulseNet
PFGE protocols, BioNumerics and the PulseNet masterscripts.

Appendix PNQ02-5

Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Campylobacter jejuni

Cultures

Biological Safety Warning: Campylobacter jejuni strains are considered Level 2 biological agents by the U.S.
Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out
laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.

Note: Store the lyophilized cultures at 4oC in the dark until they are reconstituted.

Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop

Reagents Needed:
BHI agar + 5% Rabbit Blood or equivalent Campylobacter plating media
Sterile grade reagent water
70% isopropyl alcohol

Procedure for Reviving Cultures:

Day 1

1. Document the isolate number(s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.

2. Re-suspend the lyophilized cells with 250 μl of sterile grade reagent water. Allow to stand for a few minutes
and/or mix gently to produce a uniform suspension. Pipet 100µl of Campylobacter cell suspension onto a blood agar
plate (BAP) and with a 10µl loop streak for growth. Incubate at 37ºC for 48 hours.

Days 2 and 3

1. Check the plate; if the culture appears pure, pick an isolated colony, and inoculate a fresh plate for heavy growth;
incubate microaerobically for 24 hours at 37oC. Use the growth from this plate to make PFGE plugs of the C. jejuni
strains. Before making the plugs, transfer culture to fresh medium and incubate microaerobically at 37oC for 24
hours; this will ensure that the same culture can be retested, if necessary.

After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let me know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze
(-70°C) or stock these strains according to your laboratory’s policy within 1 week of receiving them. Then

VERSION: REPLACED BY: AUTHORIZED BY:

Page 16 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

your laboratory will have stock cultures of this PulseNet certification set for future use, including the
PulseNet certification of additional personnel.

The strain numbers of the Campylobacter cultures are as follows:

D424 D445 D2261 D2579

Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Campylobacter
jejuni by Pulsed Field Gel Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL03) for detailed instructions for
making the Campylobacter PFGE plugs and the “One-Day (24-28 h) Standardized Laboratory Protocol for
Molecular Subtyping of Escherichia coli, Salmonella serotypes and Shigella sonnei by Pulsed Field Gel
Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL05) to make the H9812 standard plugs.
Both sections are available in the PulseNet QA/QC Manual. This document is available in the QA/QC conference
of the CDC Team Support or at www.cdc.gov/pulsenet/. If you can not access the protocol, please request it by
sending an E-mail to [email protected].

1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.
2. Restrict one plug slice from the four test cultures (D424, D445, D2261, and D2579) with SmaI for
2 - 4 hours at 25°C and 2 plug slices of Salmonella ser. Braenderup H9812 standard with XbaI for
at least 2 hours at 37°C.
3. Load H9812 in Lanes 1 and 6 and the test strains as follows:

Lane Certification Strain Enzyme

1 H9812 XbaI
2 D424 SmaI
3 D445 SmaI
4 D2261 SmaI
5 D2579 SmaI
6 H9812 XbaI

Note: If you want to use combs with 15 teeth, load the plug slices in lanes 3-9 and leave the other lanes empty. (The
use of the ~0.5-mm wide 15-teeth comb [Bio-Rad, 170-4326] in the standard casting stand [14 x 13-cm] is not
recommended for routine PFGE analysis of test isolates, but its use will be allowed for certification.).

4. Run the gel using the Campylobacter jejuni electrophoresis conditions. The run times listed in the
protocol are based on the equipment and reagents used at CDC. If the gels generated in your lab
do not have the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel,
the run time may have to be changed.

5. Staining, de-staining, and gel documentation (imaging):

a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns that will automatically
be asked to rerun the gel and submit a new certification TIFF. Images that have a file size that
is not within the range of ~300-400 kb will be deemed unsatisfactory and certification file
evaluators will ask for a resubmission.

b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before

VERSION: REPLACED BY: AUTHORIZED BY:

Page 17 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

restriction is repeated. If the problem persist repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme.

7. Bundle file creation

a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database
entries (the 4 test strains) linked with SmaI.
b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines
PND04 for instructions on how to create a PulseNet Bundle File). Bundles created using this
icon are “PulseNet bundles” and will contain only standardized PulseNet experiments and
fields. “PulseNet bundles” can be recognized by the “PN” that is automatically added to the
file name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.

8. Send the TIFF file and/or bundle (*.bdl) file of your gel images to the CDC Pulse Net Database
Administrator at [email protected] within four weeks after receiving the strains.
a. In the email to CDC, include Campylobacter Certification in the Subject line, so it can be
forwarded to the correct person. Include the isolate number and restriction enzyme used for
each lane on the gel in the body of the email. If you are sending certification TIFFs and
bundle files for more than one organism, please send one email per organism.
b. Name the TIFF and bundle files of your gel images as follows:
TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to
four letters of the file. The next 2 spaces will indicate the year the file was created. The next
3 spaces indicate the sequential number of the file submitted from your laboratory during a
calendar year. For example: GA09012.tif is the twelfth file submitted from the state of
Georgia during 2009. Bundle: The bundle files are named in a similar manner as the TIFF
files with the first two to four letters of the file name indicating the unique identifier code.
The next 2 spaces indicate the year the bundle file was created, and the next 3 spaces indicate
the sequential bundle file number from each laboratory. For example, the eighth bundle file
submitted from the state of Georgia during 2009 would be named GA09008PN.bdl.

For each PulseNet pathogen, an individual may be certified for:

a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.

After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 18 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

Appendix PNQ02-6

Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Vibrio cholerae Cultures

This package contains lyophilized cultures of the four Vibrio cholerae strains that are used for PulseNet
Certification. After the strains have been reconstituted according to the enclosed directions, streak each culture to
agar plates, pick an isolated colony, and subculture to another plate. Use the growth from the second plate to make
the PFGE plugs. Please let us know if the package does not arrive in satisfactory condition, or if the cultures are not
viable. Freeze (-70°C) or stock these strains according to your laboratory’s policy using 24 – 48 h growth
within one week after reconstituting them. Then, your laboratory will have stock cultures of this PulseNet
certification set for future use, including the PulseNet certification of additional personnel.

The strain numbers of the Vibrio cultures are as follows:

CDC-100 CDC-104 CDC-105 CDC-106

Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Vibrio cholerae by
Pulsed Field Gel Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL06) for detailed instructions for making
the Vibrio PFGE plugs and the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of
Escherichia coli, Salmonella serotypes and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE)” (PulseNet
QA/QC Manual PNL05) to make the H9812 standard plugs. Both sections are available in the PulseNet QA/QC
Manual. This document is available in the QA/QC conference on CDC Team or at www.cdc.gov/pulsenet/. If you
can not access the protocol, please request it by sending an E-mail to [email protected].
1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they can be
retested several times, if necessary.
2. Restrict one plug slice from the four test cultures (CDC-100, CDC-104, CDC-105, and CDC-106) with
SfiI for 4 hours at 50°C and 3 plug slices of Salmonella ser. Braenderup H9812 standard with XbaI for
at least 2 hours at 37°C.
3. Restrict one plug slice from the three test cultures (CDC-104, CDC-105, and CDC-106) with NotI for 4
hours at 37°C.
4. Load H9812 in Lanes 1, 6, 10 and the test strains as follows:

Lane Certification Strain Enzyme

1 H9812 XbaI
2 CDC-100 SfiI
3 CDC-104 SfiI
4 CDC-105 SfiI
5 CDC-106 SfiI
6 H9812 XbaI
7 CDC-104 NotI
8 CDC-105 NotI
9 CDC-106 NotI
10 H9812 XbaI

Note: If you want to use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty.
(The use of the ~0.5-mm wide 15-teeth comb [Bio-Rad, 170-4326] in the standard casting stand [14 x 13-cm] is not
recommended for routine PFGE analysis of test isolates, but its use will be allowed for certification.).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 19 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

5. Run the gel using the V. cholerae electrophoresis conditions. The run times listed in the protocol
are based on the equipment and reagents used at CDC. If the gels generated in your lab do not
have the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run
time may have to be changed.

6. Staining, de-staining, and gel documentation (imaging):

a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns that will automatically
be asked to rerun the gel and submit a new certification TIFF. Images that have a file size that
is not within the range of ~300-400 kb will be deemed unsatisfactory and certification file
evaluators will ask for a resubmission.

b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persist repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme.

6. Bundle file creation

a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database entries
as follows: 3 test strains restricted with SfiI and NotI and one test strain restricted only with SfiI.
If there are more than 4 entries in the bundle file, one or more lanes have not been linked
properly.

b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines
PND04 for instructions on how to create a PulseNet Bundle File). Bundles created using this
icon are “PulseNet bundles” and will contain only standardized PulseNet experiments and
fields. “PulseNet bundles” can be recognized by the “PN” that is automatically added to the file
name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.

7. Send the TIFF file and/or bundle (*.bdl) file of your gel image to CDC at [email protected] within four
weeks after receiving the strains.

a. In the email to CDC, include Vcholerae Certification in the Subject line, so it can be
forwarded to the correct person. Include the isolate number and restriction enzyme used for each
lane on the gel in the body of the email. If you are sending certification TIFFs and bundle files for
more than one organism, please send one email per organism.

b. Name the TIFF and bundle files of your gel images as follows:

TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to
four letters of the file. The next 2 spaces will indicate the year the file was created. The
next 3 spaces indicate the sequential number of the file submitted from your laboratory
during a calendar year. For example: GA09012.tif is the twelfth file submitted from the
Georgia Public Health Laboratory during 2009.

Bundle: The bundle files are named in a similar manner as the TIFF files with the first two
to four letters of the file name indicating the unique identifier code. The next 2 spaces
indicate the year the bundle file was created, and the next 3 spaces indicate the sequential
VERSION: REPLACED BY: AUTHORIZED BY:

Page 20 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

bundle file number from each laboratory. For example, the eighth bundle file submitted
from the Georgia Public Health Laboratory during 2009 would be named GA09008PN.bdl.
Remember, “PN” is automatically added to the file name.

For each PulseNet pathogen, an individual may be certified for:

a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.

After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory.

If the TIFF images or bundle files are not satisfactory, the submitter will need to review the troubleshooting
comments received from the evaluator and resubmit once results have improved. If the submitter fails certification
three times, the individual will not be allowed to submit again for six months. Before resubmitting, the individual
will be expected to work with CDC and/or their PulseNet Area Laboratory until satisfactory results are achieved.
This includes, but is not limited to, troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and
the PulseNet Master Scripts.

Appendix PNQ02-7

Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Vibrio parahaemolyticus

Cultures

Biological Safety Warning: Vibrio parahaemolyticus strains are considered Level 2 biological agents by the U.S.
Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry
out laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.

Note: Store the lyophilized cultures at 4°C in the dark until they are reconstituted.

Materials Needed:
Sterile sturdy forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop

Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water or Trypticase Soy Broth (TSB)
70% isopropyl alcohol

VERSION: REPLACED BY: AUTHORIZED BY:

Page 21 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

Procedure for Reviving Cultures:

Day 1

1. Document the isolate number (s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.

2. Re-suspend the lyophilized cells with 1.0 ml of sterile grade reagent water. Allow to stand for a few minutes
and/or mix gently to produce a uniform suspension. Pipet 100µl of cell suspension onto a blood agar plate (BAP)
and with a 10µl loop streak for growth. Incubate at 37ºC overnight.

Days 2 and 3

1. Check the BAP; if the culture appears pure, pick an isolated colony, and inoculate a fresh BAP for heavy growth;
incubate at 37°C overnight. Use the growth from this plate to make PFGE plugs of the standard strains. Before
making the plugs, transfer culture to fresh medium and incubate at 37°C overnight; this will ensure that the same
culture can be retested, if necessary.

After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let us know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze (-
70°C) or stock these strains according to your laboratory’s policy using 24 – 48 h growth within one week
after reconstituting them. Then, your laboratory will have stock cultures of this PulseNet certification set for
future use, including the PulseNet certification of additional personnel.

The strain numbers of the Vibrio cultures are as follows:

CDC#200-07 CDC#201-07 CDC#202-07 CDC#203-07

Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Vibrio
paraheamolyticus by Pulsed Field Gel Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL22) for detailed
instructions for making the Vibrio PFGE plugs and the “One-Day (24-28 h) Standardized Laboratory Protocol for
Molecular Subtyping of Escherichia coli, Salmonella serotypes and Shigella sonnei by Pulsed Field Gel
Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL05) to make the H9812 standard plugs. Both sections are
available in the PulseNet QA/QC Manual. This document is available in the QA/QC conference on CDC Team or at
www.cdc.gov/pulsenet/. If you can not access the protocol, please request it by sending an E-mail to
[email protected].
1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.
2. Restrict one plug slice from the four test cultures (CDC#200-07, CDC#201-07, CDC#202-07,
CDC#203-07) with SfiI for 4 hours at 50°C and 3 plug slices of Salmonella ser. Braenderup
H9812 standard with XbaI for at least 2 hours at 37°C.
3. Restrict one plug slice from three of the three test cultures (CDC#200-07, CDC#201-07, and
CDC#202-07) with NotI for 4 hours at 37°C.
4. Load H9812 in Lanes 1, 6, 10 and the test strains as follows:

Lane Certification Strain Enzyme

1 H9812 XbaI

VERSION: REPLACED BY: AUTHORIZED BY:

Page 22 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

2 CDC#200-07 SfiI
3 CDC#201-07 SfiI
4 CDC#202-07 SfiI
5 CDC#203-07 SfiI
6 H9812 XbaI
7 CDC#200-07 NotI
8 CDC#201-07 NotI
9 CDC#202-07 NotI
10 H9812 XbaI

Note: If you want to use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty.
(The use of the ~0.5-mm wide 15-teeth comb [Bio-Rad, 170-4326] in the standard casting stand [14 x 13-cm] is not
recommended for routine PFGE analysis of test isolates, but its use will be allowed for certification.).

5. Run the gel using the V. paraheamolyticus electrophoresis conditions. The run times listed in the
protocol are based on the equipment and reagents used at CDC. If the gels generated in your lab do not
have the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run time
may have to be changed.

6. Staining, de-staining, and gel documentation (imaging):

a. The gel image should fill the entire window of the imaging equipment (computer) screen (without
cutting off wells or lower bands). Individuals submitting certification TIFFs that do not contain the
wells or clearly show the bottom bands of the patterns that will automatically be asked to rerun the
gel and submit a new certification TIFF. Images that have a file size that is not within the range of
~300-400 kb will be deemed unsatisfactory and certification file evaluators will ask for a
resubmission.

b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more shadow
bands (indicating incomplete restriction) should not be submitted for certification. To eliminate
incomplete restriction wash the plugs at least two more times with TE Buffer before restriction is
repeated. If the problem persists repeat the restriction with more units of enzyme, for a longer
amount of time and/or with a different lot of enzyme.

7. Bundle file creation

a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC strain
number in the BioNumerics “Key” field. The bundle file should contain 4 database entries as
follows: 3 test strains restricted with SfiI and NotI and one test strain restricted only with SfiI. If
there are more than 4 entries in the bundle file, one or more lanes have not been linked properly.
b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines PND04
for instructions on how to create a PulseNet Bundle File.). Bundles created using this icon are
“PulseNet bundles” and will contain only standardized PulseNet experiments and fields.
“PulseNet bundles” can be recognized by the “PN” that is automatically added to the file name. If
a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.

8. Send the TIFF file and/or bundle (*.bdl) file of your gel image to CDC at [email protected] within four
weeks after receiving the strains.

a. In the email to CDC, include Vparahaem Certification in the Subject line, so it can be forwarded
to the correct person. Include the isolate number and restriction enzyme used for each lane on the
gel in the body of the email. If you are sending certification TIFFs and bundle files for more than
one organism, please send one email per organism.
b. Name the TIFF and bundle files of your gel images as follows:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 23 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

TIFF: Use the unique laboratory identifier code that was assigned by CDC PulseNet for the first
two to four letters of the file. The next 2 spaces will indicate the year the file was created. The next
3 spaces indicate the sequential number of the file submitted from your laboratory during a calendar
year. For example: GA09012.tif is the twelfth file submitted from the Georgia Public Health
Laboratory during 2009.
Bundle: The bundle files are named in a similar manner as the TIFF files with the first two to four
letters of the file name indicating the unique identifier code. The next 2 spaces indicate the year the
bundle file was created, and the next 3 spaces indicate the sequential bundle file number from each
laboratory. For example, the eighth bundle file submitted from the Georgia Public Health
Laboratory during 2009 would be named GA09008PN.bdl. Remember, “PN” is automatically
added to the file name.

For each PulseNet pathogen, an individual may be certified for:

a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.

After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the TIFF images or bundle files are not
satisfactory, the submitter will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet Master Scripts.

Appendix PNQ02-8

Analysis Certification Protocol for E. coli Non O157 (STEC)

PulseNet participants may become analysis-certified for E. coli Non O157 (STEC) by analyzing a TIFF
sent by CDC (KC09104.tif) and creating a PulseNet bundle file. There must be at least one person in your
laboratory that is currently TIFF-certified in E. coli O157:H7 before anyone else from your laboratory
becomes analysis-certified for E. coli Non O157.

1. Lane Information for KC09104.tif.

Note 1: If more than one person in your laboratory will be submitting this certification set, you may wish to
rename the TIFF using your initials (i.e. JKA09104.tif or KC09104JKA.tif) before you import it into your
local database (otherwise the TIFF will be overwritten each time it is imported by a different person).

Note 2: When assigning reference markers in step 3 (normalization) of analysis place the marker at the top
of the band at 668.9 Kb and the top band of the doublet at 167.1 Kb as the reference standard in the
database suggests.

Lane Certification Strain Enzyme

1 H9812 XbaI

VERSION: REPLACED BY: AUTHORIZED BY:

Page 24 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

2 3074-01 XbaI
3 3508-03 XbaI
4 K5159 XbaI
5 K4947 XbaI
6 H9812 XbaI
7 3074-01 BlnI
8 3508-03 BlnI
9 K5159 BlnI
10 H9812 XbaI

2. Bundle File Creation

a. In the bundle file enter the corresponding CDC strain number in the BioNumerics “Key” field. If
there are more than 4 entries in the bundle file, one or more lanes have not been linked properly.
The standard lanes should never be linked.

b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines PND04
for instructions on how to create a PulseNet Bundle File). Bundles created using this icon are
“PulseNet bundles” and will contain only standardized PulseNet experiments and fields.
“PulseNet bundles” can be recognized by the “PN” that is automatically added to the file name. If
a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.

3. Send the bundle (*.bdl) file of your gel image to CDC at [email protected].

a. In the email to CDC, include E. coli Non O157 Certification in the Subject line, so it will be
directed to the correct person. If you are submitting certification TIFFs and bundle files for more
than one organism, please send one email per organism.

b. Name the bundle file containing your analysis as follows:

Bundle files are named in a similar manner as TIFF files with the first two to four letters of the file
name indicating your unique laboratory identifier code. The next 2 spaces indicate the year the
bundle file was created, and the next 3 spaces indicate the sequential bundle file number from each
laboratory. For example, the eighth bundle file submitted from the Georgia Public Health
Laboratory during 2009 would be named GA09008PN.bdl. Remember, “PN” is automatically
added to the file name.

After your analysis bundle file is submitted, the PulseNet certification evaluator will analyze your submission and
inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the
files. If the bundle file is satisfactory, the person who submitted the bundle file will be issued a SecurID device (also
called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID devices are
issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if the certified
individual leaves his or her position in the laboratory. If the bundle file is not satisfactory, the submitter will need to
review the troubleshooting comments received from the evaluator and resubmit once results have improved. If the
submitter fails certification three times, the individual will not be allowed to submit again for six months. Before
resubmitting, the individual will be expected to work with CDC and/or their PulseNet Area Laboratory until
satisfactory results are achieved. This includes, but is not limited to, troubleshooting and training in the PulseNet
protocols, BioNumerics and the PulseNet Master Scripts.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 25 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

Appendix PNQ02-9

Analysis Certification Protocol for C. botulinum

This package contains cultures of four Clostridium botulinum strains used for PulseNet Certification. Since this is a
Select Agent transfer, your Responsible Official (RO) must complete Section 3 of APHIS/CDC Form 2 and send a
copy to APHIS/CDC within two business days of receipt. If the package has been damaged to the extent that a
release of select agent may have occurred, your laboratory’s RO must immediately report to APHIS/CDC. Please
refer to National Select Agent Registry website (http://www.selectagents.gov/TransferForm.html) for additional
information.

Stock these four C. botulinum strains according to your laboratory’s policy within one week after receiving them.
Then, your laboratory will have stock cultures of this PulseNet certification set for future use, including the PulseNet
certification of additional personnel.

The strain numbers of the C. botulinum cultures are as follows:

CDC5328 CDC44575 CDC37456 CDC55707

Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “Five Day Standardized Laboratory Protocol for Molecular Subtyping of Clostridium botulinum
by Pulsed Field Gel Electrophoresis (PFGE). This document is available as PNL25 in the QA/QC Manual
conference on CDC Team or at www.cdc.gov/pulsenet/. If you cannot access the protocol, please request it by
sending an E-mail to [email protected].

1. Streak each test culture for colony isolation onto EYA plates. Record isolate number and date on each
plate. Incubate under anaerobic conditions at 37°C (± 2) for two days.

2. If the culture appears pure, pick an isolated colony and streak for colony isolation onto ANA-BAP plates.
Incubate under anaerobic conditions overnight at 37°C ± 2°C.

3. Make two or three disposable plug molds plugs, or two reusable plug molds plugs of each test strain so they
can be retested several times, if necessary.

4. Restrict three plug slices of Salmonella ser. Braenderup H9812 (the PulseNet Universal Standard Strain)
with XbaI (60 Units/plug slice) for 2 hours at 37°C.

5. Restrict one plug slice from the four test cultures (CDC5328, CDC44575, CDC37456, and CDC55707)
with SmaI (50 Units/plug slice) for 4 hours at 25°C.

6. Restrict one plug slice from three of the test cultures (CDC5328, CDC44575, and CDC37456) with XhoI
(100 Units/plug slice) for 3 hours at 37oC.

7. Load H9812 and the test strains as follows:

Lane Certification Strain Enzyme

1 H9812 XbaI
2 CDC5328 SmaI
3 CDC44575 SmaI
4 CDC37456 SmaI
5 H9812 XbaI

VERSION: REPLACED BY: AUTHORIZED BY:

Page 26 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

6 CDC5328 XhoI
7 CDC44575 XhoI
8 CDC37456 XhoI
9 CDC55707 SmaI
10 H9812 XbaI

Note: If you use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty
(Although the use of the ~0.5-mm wide 15-teeth combs [Bio-Rad, 170-4326] in the standard casting stand
[14 x 13-cm] is not recommended for routine PFGE analysis of test isolates, the use of the smaller comb
teeth will be allowed for certification.).

8. Run the gel using the C. botulinum electrophoresis conditions. These run times are based on the equipment
and reagents used at CDC. If the gels generated in your lab do not have the lowest band in strain H9812
approximately 1-1.5 cm from the bottom of the gel, the run time may have to be changed.

9. Staining, de-staining, and gel documentation (imaging):

a. The gel image should fill the entire window of the imaging equipment (computer) screen (without
cutting off wells or the bottom of the gel). An individual submitting a certification TIFF with wells
cut off or bottom bands that are not visible will automatically be asked to rerun the gel and submit
a new certification TIFF. Images that have a file size that is not within the range of ~300-400 kb
will be deemed unsatisfactory and certification file evaluators will ask for a resubmission.

b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more shadow
bands (indicating incomplete restriction) should not be submitted for certification. To eliminate
incomplete restriction wash the plugs at least two more times with TE Buffer before restriction is
repeated. If the problem persists, repeat the restriction with more units of enzyme, for a longer
amount of time and/or with a different lot of enzyme.

10. Bundle file creation

a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC strain
number in the BioNumerics “Key” field. The bundle file should contain 4 database entries as
follows: 3 test strains restricted with SmaI and XhoI and one test strain restricted only with SmaI.
If there are more than 4 entries in the bundle file, one or more lanes have not been linked properly.

b Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines PND04
for instructions on how to create a PulseNet Bundle File). Bundles created using this icon are
“PulseNet bundles” and will contain only standardized PulseNet experiments and fields.
“PulseNet bundles” can be recognized by the “PN” that is automatically added to the file name. If
a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.

11. Send the TIFF file and/or bundle (*.bdl) file of your gel images to CDC PulseNet at [email protected]
within four weeks after receiving the strains.

a. In the email to CDC, include C. botulinum Certification in the Subject line, so it can be
forwarded to the correct person. Include the isolate number and restriction enzyme used for each
lane on the gel in the body of the email.

b. Name the TIFF and bundle files of your gel images as follows:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 27 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two or three
letters of the file. The next 2 spaces will indicate the year the file was submitted. The next 3 spaces
indicate the sequential number of the file submitted from your laboratory during a calendar year. For
example: GA10012.tif is the twelfth file submitted from the state of Georgia during 2010.

Bundle: The bundle files are named in a similar manner as the TIFF files with the first 2 or three letters
of the file name indicating the unique identifier code for your laboratory. The next 2 spaces indicate
the year the bundle file was submitted, and the next 3 spaces indicate the sequential bundle file number
from each laboratory. For example, the eighth bundle file submitted from the state of Georgia during
2010 would be named GA10008PN.bdl.

For each PulseNet pathogen, an individual may be certified for:

a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.

After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.

Appendix PNQ02-10

Shigella flexneri Analysis Certification

PulseNet participants may become analysis-certified for S. flexneri by analyzing a TIFF sent by
CDC (MC14025.tif) and creating a PulseNet bundle file. There must be at least one person in
your laboratory that is currently gel and analysis-certified in Shigella sp. before anyone else from
your laboratory becomes analysis-certified for Shigella flexneri.

1. Lane Information for MC14025.tif.

Note 1: If more than one person in your laboratory will be submitting this certification set, you
may wish to rename the TIFF using your initials (i.e. DS14025I.tif or MC14025DS.tif) before
you import it into your local database (otherwise the TIFF will be overwritten each time it is
imported by a different person).

VERSION: REPLACED BY: AUTHORIZED BY:

Page 28 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

Lane Certification Enzyme

Strain
1 H9812 XbaI
2 AM46721 NotI
3 AM48331 NotI
4 2013c-3571 NotI
5 2012AM-0435 NotI
6 H9812 XbaI
7 AM46721 XbaI
8 AM48331 XbaI
9 2013c-3571 XbaI
10 H9812 XbaI

2. Bundle File Creation

a. In the bundle file enter the corresponding CDC strain number in the BioNumerics “Key”
field. If there are more than 4 entries in the bundle file, one or more lanes have not been
linked properly. The standard lanes should never be linked.

b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines
PND04 for instructions on how to create a PulseNet Bundle File). Bundles created using
this icon are “PulseNet bundles” and will contain only standardized PulseNet experiments
and fields. “PulseNet bundles” can be recognized by the “PN” that is automatically added
to the file name. If a non-PulseNet bundle is submitted, you will automatically be asked to
resubmit.

3. Send the bundle (*.bdl) file of your gel image to CDC at [email protected].

a. In the email to CDC, include Shigella Flexneri Certification in the Subject line, so it will
be directed to the correct person. If you are submitting certification TIFFs and bundle files
for more than one organism, please send one email per organism.

b. Name the bundle file containing your analysis as follows:

Bundle files are named in a similar manner as TIFF files with the first two to four letters
of the file name indicating your unique laboratory identifier code. The next 2 spaces
indicate the year the bundle file was created, and the next 3 spaces indicate the sequential
bundle file number from each laboratory. For example, the eighth bundle file submitted
from the Georgia Public Health Laboratory during 2009 would be named
GA09008PN.bdl. Remember, “PN” is automatically added to the file name. If you find
yourself attempting to add this extension, you have not created the bundle file correctly.

After your analysis bundle file is submitted, the PulseNet certification evaluator will analyze your
submission and inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within
four weeks of receiving the files. If the bundle file is satisfactory, the person who submitted the bundle
file will be issued a SecurID device (also called a fob) and will be granted on-line access to the
appropriate PulseNet national database. SecurID devices are issued to individuals within a laboratory.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 29 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ02
Effective Date:
PFGE CERTIFICATION OF PULSENET PERSONNEL 01 23 14

The devices cannot be shared and must be returned to CDC if the certified individual leaves his or her
position in the laboratory. If the bundle file is not satisfactory, the submitter will need to review the
troubleshooting comments received from the evaluator and resubmit once results have improved. If the
submitter fails certification three times, the individual will not be allowed to submit again for six months.
Before resubmitting, the individual will be expected to work with CDC and/or their PulseNet Area
Laboratory until satisfactory results are achieved. This includes, but is not limited to, troubleshooting and
training in the PulseNet protocols, BioNumerics and the PulseNet Master Scripts.

Please let us know if you have questions or further clarification is needed.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 30 of 30
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

1. PURPOSE: To describe the procedure for evaluating certification files sent by PulseNet participating
laboratories. This is part of the PulseNet QA/QC program.

2. SCOPE: This procedure applies to PulseNet personnel who evaluate certification files and certify individuals
for gels (laboratory methods for PFGE and image acquisition) and/or BioNumerics gel analysis. It also applies
to individuals who review certification reports.

3. DEFINITIONS/TERMS:

3.1 Certification files: TIFF and/or bundle files submitted by PulseNet participating laboratories for certification
evaluation.
3.2 QA/QC: Quality Assurance/Quality Control
3.3 PFGE: Pulsed-field Gel Electrophoresis
3.4 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
3.5 SOP: Standard Operating Procedure
3.6 TIFF: Tagged Image File Format, a file of a gel image that can be analyzed in BioNumerics
3.7 Certification file evaluator: An individual who evaluates certification files
3.8 TIFF quality: The grading of the appearance and ease of analysis of a TIFF according to the PulseNet TIFF
Grading Guidelines. This is a main component of the evaluation of a TIFF submitted for certification
3.9 Gel analysis assessment: The grading of the whole analysis of a TIFF, including gel and lane definition,
normalization, and band marking, according to the PulseNet Gel Analysis Guidelines. This is a main
component of the evaluation of a bundle file submitted for certification
3.10 Bundle file: A file with a .bdl extension that is produced in BioNumerics and contains the analysis of at least
one lane of a gel image
3.11 CDC: Centers for Disease Control and Prevention
3.12 Certification file reviewer: An individual who reviews and signs off on the certification reports submitted by
the certification file evaluator
3.13 Comparison TIFFs: One or more TIFFs run by CDC for a specific pathogen for use in comparing PFGE
patterns and band resolution against submitted certification TIFFs. Comparison TIFFs can also be a group of
certification TIFFs submitted by several laboratories to monitor PFGE patterns and band resolution over
several submitting laboratories. The latter is most easily accomplished through a saved list in BioNumerics
3.14 Comparison list: A list of analyzed lanes from comparison TIFFs that is saved in BioNumerics and used to
compare to the analysis of the submitted TIFF by the certification file evaluator and the analysis of the
submitter in the certification bundle file
3.15 Gel certified: Formerly “TIFF certified,” an individual or laboratory that is certified in laboratory methods for
PFGE and image acquisition
3.16 Analysis certified: An individual who is certified in BioNumerics gel analysis

4. RESPONSIBILITIES

4.1 Individuals performing PulseNet-related work (i.e., preparing PFGE gels and/or analyzing TIFF images) must
submit certification file(s) and have them evaluated before being allowed to submit TIFF images and
BioNumerics analyses directly to the PulseNet national databases. See PNQ02 for information on how to
request certification sets.

4.2 Individuals evaluating certification files (evaluators) must:

4.2.1 Assess the TIFF quality and the ease of analysis of the TIFFs submitted for certification
and assess the gel analysis, including band marking, of bundle files submitted for certification. All
files submitted should be evaluated based on the current PulseNet standards of TIFF quality and gel
analysis.
4.2.2 Evaluate submitted files and submit a written report, using the existing templates, to
CDC reviewers within the time frame written in the certification instructions sent to the participating
laboratories with the certification strain sets.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

4.2.3 Modify reports based on CDC reviewers’ comments, if necessary. Resubmit reports to
CDC as soon as possible.
4.2.4 Keep electronic files in an organized folder system and keep hard copies of TIFFs,
worksheets, reports, and any analyses (e.g., dendrograms, lane-to-lane comparisons) generated in files
organized by laboratory.

4.3 Individuals reviewing certification reports submitted by the evaluator (reviewers) must:
4.3.1 Review submitted reports and their associated cover letters and certificates within the
time frame written in the certification instructions sent to the participating laboratories with the
certification strain sets.
4.3.2 Submit signed reports to the next reviewer or to the Database Team Leader (Kelley
Hise).

5. PROCEDURE
5.1 TIFF and/or bundle files are received via email to the PFGE inbox are moved into the organism pending
subfolder within the QA/QC folder. The person assigned to evaluate that organisms’ certification file will
retrieve the submissions from this location.
5.2 The email is printed and the attached files are saved to a folder on the hard drive (or network drive) that
indicates which pathogen, which laboratory submitted the files, and which person in the laboratory submitted
the files (P:\QAQC\Certification\Certification-Final Reports\organism\lab ID-initials of submitter).
5.3 The file names and the date received are recorded in the appropriate Excel sheet (e.g., Salmonella Certification
Status_Steward.xls). The Excel sheets are saved to P:\QAQC\Certification\Certification Tables and updated
regularly.
5.4 When ready to analyze, open the TIFF in an image program. Invert to black bands on a white background, if not
submitted this way. Print out the TIFF picture, filling the whole 8 ½ x 11” sheet with the image, if possible.
5.5 Grade the TIFF visually; using the TIFF Grading Guidelines and also compare PFGE patterns to hard copies of
comparison TIFF(s).
5.5.1 If the TIFF is not passable (e.g., too much debris, bands not resolved well enough for
analysis, one or more PFGE patterns are not correct), email the sender to repeat the TIFF. Give
specific instructions about how to improve the next gel. Write on the printed email that you requested a
repeat submission and file in your working folder. Record repeat submission request on the appropriate
Excel sheet.
5.5.2 If the TIFF is passable, perform analysis as outlined in 5.6 below.
5.6 Analysis of certification TIFFs
5.6.1 Fill out the top third of a log-in and analysis sheet (login & analysis_worksheet.doc),
recording information about the current certification files and about any previous certification files
submitted but repeated.
5.6.2 In the middle third of the log-in and analysis sheet, record information about the gel
quality. Use the PulseNet TIFF Grading Guidelines, circling a rating for each category listed (4 being
the highest rating and 1 being the lowest). To the right of each category, record specific information
pertaining to that rating (e.g., Bands: clear & distinct? 2 “Bands very fuzzy and difficult to analyze in
some places”.)
5.6.3 In the bottom third of the log-in and analysis sheet, record information about the
following:
5.6.3.1 Standards – Are the standards in the correct lanes? Are the patterns correct and
distinguishable according to the current PulseNet standards and comparison TIFF(s)?
5.6.3.2 TIFF: “By eye”-match comparison gel? – Are the PFGE patterns of the test lanes
correct and distinguishable according to the current PulseNet standards and comparison
TIFF(s)?
5.6.4 Analyze the TIFF in BioNumerics. Analyze all 10 lanes of the TIFF (standard and test
lanes). Code each “key” entry with the Laboratory ID, the submitter’s initials, and the strain number
(e.g., CDC-KH-H9812).
5.6.4.1 If no bundle file was submitted, perform a dendrogram match with the comparison
VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

list (the Salmonella list is named “2003_cert”; the E. coli list is named “2003comp” and the
Listeria list is named “Compcert”) using PulseNet standard settings. Proceed to step 5.6.6 of
this document.
5.6.4.2 If a bundle file was submitted, go to step 5.6.5 below.
5.6.5 Bundle file analysis
5.6.5.1 Highlight the existing bundle file in BioNumerics and check the information before
opening. Note whether there are non-PulseNet standardized fields and/or experiments in the
bundle file. Make sure all entries are in the bundle file and linked correctly. If the lanes are
linked incorrectly or if there are no standards in the bundle file, send an email to request a
repeat submission, noting any other changes that need to be made in the repeat submission.
5.6.5.2 Open the bundle file and select the lanes (noting if the lanes are linked incorrectly).
5.6.5.3 Using PulseNet standard settings, perform a dendrogram match of the comparison
list, the bundle file analysis, and the analysis of the TIFF by the evaluator. Proceed to step
5.6.6 of this document.
5.6.6 Analysis of TIFF and/or bundle file once dendrogram is generated in BioNumerics.
5.6.6.1 Print out a hard copy of the dendrogram and go through each isolate match, circling
where the discrepancies are and looking at the submitted TIFF to see if that TIFF has a
different band resolution than the comparison.
5.6.6.2 Underneath the dendrogram, record information for each isolate with a discrepancy.
For example:
5.6.6.2.1 “A single band is resolved at ~235 kb. Some labs are resolving a doublet at this position.”
If some laboratories are resolving a particular area as a single band and some as a doublet,
this is passable. If all labs are resolving a particular area as a doublet and the certification
TIFF shows a thick single band, the TIFF should be repeated (do not pass the TIFF or
bundle file for certification), especially if this occurs in more than one place on the TIFF.
5.6.6.2.2 “A doublet is marked on the bundle file at ~400 kb that appears as a single band on the
TIFF.” – If a laboratory “over-marks” several areas of the TIFF, request a repeat bundle
file.
5.6.6.2.3 “A doublet is marked on the bundle file at ~170 kb. It is difficult to distinguish a doublet
from a single band at ~170 kb on the TIFF.” – Use this comment when bands are fuzzy
and difficult to distinguish in a certain area and the submitter has marked the area on the
bundle differently than the certification evaluator has marked it. Ask for a repeat
submission if the fuzzy bands occur over more than several places on the TIFF and the
evaluator feels that better band resolution would dramatically improve analysis.
5.6.6.2.4 “A band is marked in error at ~55 kb where no band appears on the TIFF”. – If a
laboratory marks stray bands in more than one place, request a repeat bundle file.
5.6.6.2.5 Standard lanes with bands not marked – ask for a repeat bundle file if standard bands are
not marked at all. If band(s) at ~170 kb (the bands not used for normalization) are not
marked, record standard comment about marking appropriate bands for normalization but
mark all bands during band finding.
5.6.6.3 Note any bands marked on the bundle file that consistently appear above or below
the bands marked by the evaluator. This could be due to incorrect normalization by the
submitter. Check to make sure the standard the submitter used is correct. Ask for a repeat
bundle submission if the normalization is incorrect. The submitter can be gel certified, but
not analysis certified until a new bundle file is evaluated and passed.
5.6.6.4 In the bottom third of the log-in and analysis worksheet, record information about
the dendrogram analyses:
5.6.6.4.1 TIFF: Analyzed TIFF matches comparisons? - How well does the analysis of the
submitted TIFF by the certification file evaluator match the comparison list of TIFF(s)
analysis? This should include information about the band resolution and ease of analysis
of the submitted TIFF. A note of “See dendrogram notes” in this box on the worksheet
may be sufficient.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

5.6.6.4.2 Bundle: Analyzed TIFF matches bundle? – How well does the submitter’s bundle file
analysis match the analysis of the submitted TIFF by the evaluator? This should include
information about inaccurate normalization or band marking. A note of “See dendrogram
notes” in this box on the worksheet may be sufficient. If a bundle file was not submitted,
no information should be recorded in this box.
5.6.6.4.3 Bundle: Bundle patterns match comparison patterns? – How well does the submitter’s
bundle file analysis match the patterns generated by CDC and other laboratories. The
CDC patterns are those within the list used to compare to the bundle file in the
dendrogram generated. If necessary, the certification file evaluator may perform a
dendrogram match using submissions from other laboratories to see if doublets or single
bands are being submitted in a certain area of a particular lane. If a bundle file was not
submitted, no information should be recorded in this box.
5.6.6.4.4 Report Comments – Use this box for recording information such as if the band resolution
is much better or much worse than other submissions.
5.7 After analysis, save the report template for that pathogen to the folder where the certification files
were saved (P:\QAQC\Certification\Certification-Final Reports\organism\lab ID-initials of submitter). Save
the report template using the submitter’s name (e.g., Salm 2003 cert report_KH.doc).
5.8 Complete the report
5.8.1 Type in the comments recorded on the dendrogram for each isolate with a discrepancy or other
comment.
5.8.2 Type in suggestions for improvement and additional comments at the bottom of the report. Use the
Standard Comments document (Appendix PNQ04-1) for wording and troubleshooting tips.
5.8.3 Type in information about previous submissions and the use of non-PulseNet standard fields and
experiments in the bundle file at the bottom of the comments section. See Standard Comments
document for wording.
5.8.4 Make sure the circles around the “TIFF satisfactory” and “bundle satisfactory” are correct for each
report.
5.8.5 Print out two copies of the report – one to send to CDC and one for the evaluator’s files.
5.9 After completing the report, save the cover letter template for that pathogen to the folder where the certification
files and report were saved (P:\QAQC\Certification\Certification-Final Reports\organism\lab ID-initials of
submitter). Save the cover letter template using the submitter’s name (e.g., Certification letter_DHHS
letterhead_Salm_KH.doc). Add the current date and the name of the submitter to the cover letter. Change the
check mark as appropriate. Print out one copy to send to CDC.
5.10 Open the Certification Certificates.ppt file and personalize the appropriate certificate. Print out one copy to
send to CDC.
5.11 Record the information about the completed certification on the Excel sheet (e.g., “date certified,” “date
delivered to CDC”).
5.12 Add the completed certification to a running list of all certifications completed by the evaluator. Record the
name of the submitter and his/her laboratory, whether they were certified for gel and/or analysis, the
pathogen, the date of the report (date analyzed), the date delivered to CDC, and any comments about the
analysis or the TIFF.
5.13 Paperclip the cover letter, report, and certificate together. Send to the Database Team Leader (Kelley Hise) at
CDC. The Team Leader will distribute the certifications among the Database Team for review. Send an email
to the Database Team Leader (Kelley Hise). In the email, include a list of the certifications being sent to
CDC. List the pathogen, the certified individual’s name, the laboratory ID, and what the individual was
certified for (i.e., gels and analysis, gels only, or analysis only).
5.14 Paperclip all the analyses, emails, TIFFs, etc. together with the log-in and analysis sheet on top, and file in
evaluator’s files in the appropriate laboratory folder.
5.15 Review certification files.
5.15.1 When received by CDC, the Database Team member and Methods Validation Laboratory Team
member will review the reports in a timely manner in accordance with the certification instructions
sent out with the strains to the participating laboratories.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

5.15.2 The review will consist of checking the TIFF quality assessment and the comments associated with
each analysis discrepancy. The reviewer will only sign his/her name if he/she agrees with the
evaluator’s assessment of the certification files. If the reviewer does not agree with the evaluator’s
assessment, the reviewer must contact the evaluator and discuss modifying the certification report.
5.15.3 Once the Database Team reviewer approves a certification, he or she must send an email to the
PulseNet Technical Steward listing who was certified, their lab ID, organism for which they were
certified, and what they were certified for (gels, analysis, or both).
5.16 TIFF images that pass certification and review indicate that the submitter is gel certified. Bundle files that
pass certification and review indicate that the submitter is analysis certified. The submitter is considered
certified as long as the laboratory successfully completes annual proficiency testing. If the submitted
certification files do not pass the certification evaluation, the individual will need to review the
troubleshooting comments received from the evaluator and resubmit once results have improved. If the
submitter fails certification three times, the individual will not be allowed to submit again for six months.
Before resubmitting, the individual will be expected to work with CDC and/or their PulseNet Area
Laboratory until satisfactory results are achieved. This includes, but is not limited to troubleshooting and
training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:

8.1 Database Team Leader

Kelley Hise
[email protected]
(404) 639-0704

8.2 PulseNet Technical Steward

Susan Hunter
[email protected]
(404) 639-1749

9. AMENDMENTS:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

Appendix PNQ04-1.

Standard Comments for Certification and Proficiency Testing Reports

“General” Comments:
ƒ A gel image that fills the entire window on the imaging equipment screen (without cutting off wells or the bottom of the
gel) may improve analysis.

ƒ A gel image that fills the entire window on the imaging equipment screen (without cutting off wells or the bottom of the
gel) may make analysis easier.

ƒ Lanes X-Y are skewed slightly but the skew does not interfere with the analysis.

ƒ The TIFF file was renamed <labID>051602.tif by evaluator for analysis. In the future, please name all files sent to
CDC according to the standardized PulseNet naming system. The first two digits should be the laboratory ID (e.g.,
GA), the second two digits should be the year (e.g., 04), and the last three digits should be the unique number of
the TIFF or bundle file submitted that year from your laboratory (e.g., 001).

Band Marking:
ƒ Consistency in band marking could be improved by marking thick bands as singlets unless two bands (a doublet) can be
visualized on the TIFF.

ƒ Bands down to the last band of the standard (~20 kb) should be marked. If test bands are close to the last band of the
standard by a visual check of the TIFF, mark the band. Most labs are marking the bands at ~20 kb in lanes 7 and 8.
Sometimes these bands run a little above the standard and sometimes they run a little below the standard.

ƒ Listeria 2003 (03-H8394): A singlet is marked at ~150 kb on bundle file. Some laboratories are resolving and marking 2
bands in this area. [The light band directly under the bright band at ~150 kb should be marked on the bundle file.]
[A singlet is marked at ~150 kb on bundle file. Some laboratories are resolving and marking the lighter area directly
underneath the dark band at ~150 kb as a second band.]

ƒ Listeria 2003 (04-H8395): A doublet is marked at ~50 kb on bundle file. Some laboratories are resolving and marking a
third band at ~40 kb.

Standard Lanes:
ƒ Please include standard lanes in bundle file for certification.

ƒ Please place standards in the assigned lanes described in the certification or proficiency testing protocols.

ƒ The outer two standard lanes skewed inward, but the skew did not interfere with the analysis.

ƒ Bands of some of the standard lanes (especially lanes 1 and 15) are lighter than bands of the test strains. This did not
affect analysis. However, more consistent standardization of cell concentrations among all test and standard strains
would improve gel quality.

ƒ The band distortion in the first and last lanes could cause inaccurate analysis if the band placement was not adequately
checked during normalization.

ƒ Although for normalization with standard strain H9812 the ninth band at ~180 kb is not marked, for band
finding, it should be marked as it appears on the TIFF. In this case, it should be marked as a single band.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

ƒ E. coli 2003 H9812: The area at ~170 is marked as a single band on the bundle file, but appears as a doublet on the
TIFF.

ƒ Listeria 2003: The top two bands in each standard lane are closer together than on other certification gels. Make sure
you are following the Listeria standardized protocol, especially the comb placement, for optimum band separation.

ƒ E. coli 2003: In the H9812 standard lanes, a few laboratories are resolving doublets at ~170 kb, ~76 kb, and ~33
kb such as appears in lane 5 of this TIFF. Lane 5 has very good band resolution. When doublets are resolved in
the noted areas, make sure the area of the doublet marked during normalization is consistent with the area of
any single bands at the same position on other lanes. For areas resolved as doublets but normalized as one band,
you may wish to normalize using the midpoint between the two doublet bands. However, in band finding, the
bands should be marked as they appear on the TIFF.

Gel Not Clean/Shadow Bands Present:

ƒ Bands are difficult to see on the TIFF and mark on the bundle file because the gel background is not clean. Spots and
other debris may be reduced by cleaning all staining and destaining containers and preparing fresh reagents with clean
glassware. Make sure that all agarose goes into solution when it is heated with 0.5X TBE.

ƒ Background from DNA degradation (i.e., smearing in the lanes) may be reduced by carefully preparing new plugs to
prevent shearing. Background may also occur because the agarose was too hot when the plugs were made or when the gel
was poured and/or because of poor quality reagents. Washing the plugs at least two more times with TE Buffer may also
reduce background.

ƒ “Shadow” or “ghost” bands are present. These probably indicate incomplete restriction and should not be present on a
gel. If shadow bands appear on gels in the future, repeat restriction with more units of enzyme, for a longer amount of
time and/or with a different lot of enzyme. Wash the plugs at least two more times with TE Buffer before restriction is
repeated.

ƒ The BlnI pattern of 24-98 contains an extra faint band at ~140 kb. This band has been present on certification TIFFs
received from other laboratories. This faint band may be the result of incomplete restriction or it could be due to a
mutation in the strain itself. If this certification set must be used again, repeat restriction with more units of enzyme, for a
longer amount of time and/or with a different lot of enzyme. Wash the plugs at least two more times with TE Buffer
before restriction is repeated. If that does not eliminate the faint band, you may wish to consider requesting a new 24-98
strain from CDC by emailing [email protected].

Top Bands:
ƒ Salmonella 2003: The distance between the first and second bands of the patterns on the TIFF is less than on the
comparison TIFFs. This difference may make comparison of your gels to patterns in the national database difficult,
especially when the first bands of test strain patterns are near the top of the gel. For example, the distance difference
appears to have affected the pattern of CDC 61-99 (BlnI). On the TIFF, a thick band appears at ~1070 kb for CDC
61-99 (BlnI) where a doublet appears on the comparison TIFFs. Make sure you are running the gels according to the
standardized protocol on 1% SeaKem Gold agarose (BioWhittacker) with CHEF settings of 30 Kb – 700 Kb [2.2 s –
63.8 s] for Salmonella species. You may also try running the gel for a longer period of time (without cutting off
bottom bands). Contact Mary Ann Fair ([email protected]) for help if this does not solve the problem.

Bottom Bands:
ƒ Running the gel so that the last band of the standard is approximately 1.0-1.5 cm from the bottom of the gel (per PulseNet
protocol) may improve separation of bands.

ƒ The certification was marked unsatisfactory because the bottom bands of the standard are not visible on XX.tif and are
too low on XX.tif. Please run the gel so that the last band of the standard is approximately 1.0-1.5 cm from the bottom as
per the PulseNet standard PFGE protocol.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

ƒ The bottom bands of the standard are too low. The gel should be run so that the last band of the standard is approximately
1.0-1.5 cm from the bottom per the PulseNet standard PFGE protocol.

Resolution:
ƒ This is a good TIFF but could be improved by increasing band sharpness.

ƒ Improvements in band resolution and gel background clarity will aid analysis and improve comparisons with the national
database.

ƒ Fuzzy bands may occur because of an imaging problem or because the agarose was too hot when the plugs were made or
when the gel was poured, because of incomplete plug washing, and/or because of poor quality reagents. On future gels,
you may want to check your TIFF imaging by determining if a fluorescent ruler placed beside the gel is in focus. If the
ruler is not in focus, the bands on the gel may not be either. You may want to check the filter on your GelDoc system to
make sure it is clean.

ƒ On this gel, it appears that the wells are out of focus. This may indicate that the bands are not focused. On future gels,
you may want to check your TIFF imaging by viewing the gel directly on the UV box or by determining if a fluorescent
ruler placed beside the gel is in focus. If the ruler is not in focus, the bands on the gel may not be either.

ƒ Bands seem to disappear at the bottom of the gel. This affected the bundle file analysis.

ƒ Lanes with bands missing at the bottom of the gel and/or where the pattern is light may require repeating restriction with
a larger plug slice, more units of enzyme, for a longer amount of time, and/or with a different lot of enzyme. Wash the
plugs at least two more times with TE Buffer to remove excess lysis reagents or other impurities. Preparing new plugs
with more concentrated cell suspensions may also improve band appearance. Light bands could also be the result of
prolonged exposure to UV light before capture of the image.

ƒ The bands are distinct, but, in most lanes, very light. Better standardization of cell concentrations among all test and
standard strains would improve gel quality and TIFF imaging. Lanes where the pattern is light may require repeating
restriction with a larger plug slice or preparing new plugs with more concentrated cell suspensions. Light bands could
also be the result of prolonged exposure to UV light before capture of the image. [Can add: Make sure that the UV filter
is in place while photographing the gel and that fresh ethidium bromide solution in the proper concentration (stock of 10
mg/ml diluted to 1:10,000 or 10 microliters in 100 ml reagent grade/deionized water) is used for staining gels.]

ƒ The bands tend to be very light at the bottom of the gel. Correcting this may require repeating restriction with a larger
plug slice, more units of enzyme, for a longer amount of time, and/or with a different lot of enzyme. You could also try
washing the plugs at least two more times with TE Buffer to remove excess lysis reagents or other impurities. Light bands
could also be the result of prolonged exposure to UV light before capture of the image.

ƒ This gel appears to have a different band resolution than the comparison gels; some thick single bands on the comparison
patterns appear as doublets on this gel.

ƒ Improving the resolution of the lower molecular weight bands would improve analysis.

ƒ Bands are fused, fuzzy, and difficult to distinguish. Use of a comb with 10 mm-wide teeth (instead of 5.5 mm-wide teeth)
and running the gel for a longer time may improve the separation of bands.

ƒ Better standardization of cell concentrations among all test and standard strains would improve gel quality.

ƒ In some test lanes, thick bands (possibly due to a slight DNA overload) make doublets difficult to distinguish.

ƒ There appears to be too much DNA in the lanes. Use smaller plug slices or prepare new plugs with less concentrated cell
suspensions to improve separation of bands.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

…to improve band appearance and separation for ease of analysis.

ƒ The bands appear thick. This could be the result of too much DNA in the lanes or an over-integration issue in image
capture. Use smaller plug slices or prepare new plugs with less concentrated cell suspensions to improve separation of
bands. Compare your gel as it appears on the UV box to the TIFF image. If thick bands do not appear on the gel but do
appear on the TIFF image, refer to the GelDoc manual for ways to improve your TIFF imaging (e.g., saturated pixels
function).

ƒ Listeria: Turning the comb around and running the gel so that the last band of the standard is approximately 1.0-1.5 cm
from the bottom of the gel may improve separation of bands.

ƒ Several doublets and triplets are difficult to distinguish from single bands and from doublets, respectively. For Listeria
gels, the comb is turned around so that the teeth face the top of the gel; run the gel long enough so that the last band of the
standard is approximately 1.0-1.5 cm from the bottom of the gel to help improve resolution and separation of bands.

ƒ Bands appear wavy. This could be due to one or more of the following: plug damage from the pipet tip or spatula during
removal from buffer or loading onto the gel; the comb is not clean; there are bubbles on the plug slices during loading on
a gel; the plugs are not firm or are too thin, both of which may cause increased likelihood of damage from a pipet tip.
Fragile plugs may be caused by the agarose concentration not being high enough or by overheated agarose.

ƒ The bands appear wavy. Careful handling of the plugs during plug preparation and loading may reduce distortions. Make
sure that the plug slices are firmly attached to the comb before slowly pouring the agarose into the gel mold so as not to
dislodge the plug slices.

ƒ Frowning bands could occur because of buffer flow or temperature fluctuations during the running of the gel, because the
gels sat too long before they were run, because the plugs were too thick, or for another unknown reason. Bands with more
curve than those on this TIFF would be difficult to analyze accurately.

ƒ Several lanes contain bands that are distorted. Careful handling of the plugs during plug preparation and loading may
reduce distortions.

Analysis:
ƒ Comparing your band markings in the software to a hard copy of your original TIFF may improve analysis.

ƒ Improvements in band resolution and gel background clarity will aid analysis and improve comparisons with the national
database.

ƒ During the first step of analysis in BioNumerics (1. Strips), place the top of the green box frame directly under the wells
and the bottom of the green box frame at the bottom edge of the gel. This will standardize lane sizes, which may produce
more accurate normalizations and improve comparisons with the national database.

ƒ Also during the first analysis step, using the linear adjustment under “Edit tone curve” may provide better band clarity for
analysis.

ƒ The bands in the bundle appear dark compared to the bands in the comparison lanes. This may be the result of using the
“enhance weak band” feature in the “edit tone curve” option under Step 1 of the BioNumerics analysis. Too much weak
band enhancement may make analysis more difficult. Remember to compare your band markings in the software to a
hard copy of your original TIFF.

ƒ The gel strips in XX.bdl are too wide. After the lanes are defined in BioNumerics, adjust the “thickness” under
“edit settings” to increase or decrease the thickness of the defined gel strips so that the left and right edges of the
strips are just inside the outer edge of the bands. This will ensure that the bands will appear in a size for proper

VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

analysis. If the gel strips are defined incorrectly (too wide), the resulting narrow bands with large white patches
on either side of the bands are difficult to analyze.

ƒ Do not overmark bands during analysis. If you think an area may be a doublet but no white space is apparent in
the middle of the area or no indentations appear on the sides of the area separating two bands, mark the area as a
single band or rerun a gel that has better band resolution to check the area.

ƒ To decide if an area is a doublet or single band, look on the TIFF for white space in the middle of the area or indentations
on the sides of the area separating two bands. If white space and/or indentations are present, mark the area as a doublet.

ƒ Make sure the normalization markers are placed consistently from standard lane to standard lane at the bottom of
the TIFF where doublets are resolved (~30 kb). The normalization markers should be placed on the doublet in the
same way for each standard lane: either on the top band, the bottom band or in between the 2 bands of the
doublet.

ƒ The band marking in the bundle was good but could be improved by the presence of sharper bands and more
consistency in band marking from lane to lane (e.g., marking thick bands as singlets unless two bands [a doublet]
can be visualized on the TIFF).

ƒ One extra band is marked at >2000 kb in the following lanes: Lane 5 (H2446), Lane 4 (H8395), and Lane 9
(H8395). To prevent this from happening in the future, you could do one or both of the following:
a. When first going to Step 4 Bands in BioNumerics, look at the whole gel picture (without a zoom) and
delete obviously extraneous bands. Make sure you have taken off the normalization so that all extraneous
bands have been deleted. Note - It looks like you've drawn the top of your green box outlining the gel area
(in Step 1 BioNumerics) low. Try putting the top of the green box directly under the wells next time.
b. After band finding, run a dendrogram of all the analyzed lanes. Extraneous high bands (and low bands)
will show up easily on the dendrogram.

ƒ In the bundle file, a doublet was marked at ~160 kb. This area is difficult to distinguish from a singlet on the TIFF – there
is little (if any) white area in the middle and no defined indentations on the side separating 2 bands. Most laboratories are
resolving and marking a singlet at this position.

ƒ Compare the doublet marked at ~270 kb in lane 8 (CDC 24-98, BlnI) on the bundle file to the singlet marked at
~360 kb in lane 2 (CDC 16-98, XbaI). Because the bands in the two areas look similar on this TIFF, they should
be marked consistently. When determining whether a band is a singlet or a doublet, look for white space in
between the bands and/or indentations (i.e., shoulders) on the sides of the area separating two bands.

ƒ Before linking a lane to the local database, make sure the fingerprint type (e.g., PFGE-XbaI, PFGE-BlnI, etc.) is
correct. Changing fingerprints is necessary when more than one enzyme is used for restriction on a particular gel.
If the lanes are linked with incorrect fingerprint types, possible duplicate entries in the database could occur. To
change the fingerprint type, right-click on the desired lane and select “Change fingerprint type of lane…” After
the fingerprint type is changed, you can link the lane to an entry in the database. Both fingerprint types should be
indicated with a green dot next to the one entry in the BioNumerics database.

ƒ Previous TIFF files submitted on m/d/yy (AA.tif) and on m/d/yy (BB.tif) were unsatisfactory due to a large amount of
specks present on the TIFF (AA.tif) and due to curving, distorted bands (BB.tif). The previous bundle file submitted
(BB.bdl) contained too many bands at the bottom of CDC 24-98 and did not include the standard lanes.

ƒ This report analyzes the bundle submitted mm/dd/yy. Previous bundles submitted mm/dd/yy and mm/dd/yy were
edited to delete extra bands. The auto band finding feature in BioNumerics marked many extra bands on this
TIFF. In addition, some single bands on the TIFF were marked as doublets in previous bundles.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ03
Effective Date:
THE EVALUATION OF CERTIFICATION FILES 05 09 05

ƒ This report analyzes the bundle submitted mm/dd/yy. A previous bundle submitted mm/dd/yy (AA.bdl) was
edited to mark bands at the bottom of the TIFF and to change a doublet to a single band, as it appeared on the
TIFF.

ƒ AA.tif and AA.bdl were analyzed for this report. Previous TIFFs submitted mm/dd/yy (XX.tif) and mm/dd/yy
(YY.tif) were unsatisfactory due to poor band resolution and an incorrect pattern for 61-99 (BlnI), respectively.

ƒ AA.bdl (submitted mm/dd/yy) was analyzed for this report. A previous bundle submitted mm/dd/yy (XX.bdl)
was unsatisfactory due to inaccurate band finding in the H9812 standard lanes. Lane 3 of AA.tif contains a repeat
of CDC 68-98.

ƒ AA.bdl contained fields and experiments that are not in the standardized PulseNet scripts. After installing the
December 2003 Master Scripts, create PulseNet bundle files using the bundle file lightning bolt icon on the left
side of the BioNumerics screen (PulseNet BioNumerics Version 2.5 Training Manual, December 2003, Page
104). Bundle files created using this icon will contain only PulseNet fields.

ƒ When you have edited your analysis, please create a PulseNet bundle (use the lightning bolt icon, see page 104 in
the Appendix of the Dec. 2003 CDC BioNumerics manual) and submit your new bundle (you may call it
XXb.bdl, if you wish) with your XX.tif file to [email protected], including the words “E. coli certification” in the
subject line.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 11
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

1. PURPOSE: To describe the procedure for the performance and evaluation of proficiency testing within
PulseNet. This is part of the PulseNet PFGE QA/QC program.

2. SCOPE: This procedure applies to PulseNet personnel at participating laboratories who perform proficiency
testing and to the personnel who organize the proficiency testing rounds, ship proficiency testing strains,
evaluate and review the proficiency testing submissions, and send out proficiency testing results.

3. DEFINITIONS/TERMS:

3.1 Proficiency Testing (PT): An annual assessment of the quality of the work being performed in PulseNet
participating laboratories. For each pathogen, PFGE proficiency testing includes two parts – a “TIFF sent by
CDC” and a “TIFF generated by the participating laboratory (i.e., in-house TIFF).”
3.2 QA/QC: Quality Assurance/Quality Control
3.3 PFGE: Pulsed-field Gel Electrophoresis
3.4 TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics.
3.5 Certification files: TIFF and/or bundle files submitted by PulseNet participating laboratories for certification
evaluation
3.6 SOP: Standard Operating Procedure
3.7 CDC: Centers for Disease Control and Prevention
3.8 Gel certified (or TIFF certified): An individual or laboratory that is certified in laboratory methods for PFGE
and image acquisition
3.9 “In-house TIFF”: TIFF generated by the participating laboratory. This is a part of the proficiency testing
program where laboratories run a gel and produce a TIFF of the gel that contains the Salmonella Braenderup
H9812 standards and the proficiency testing strain. The TIFF is analyzed and submitted to the organism-
specific online PT database. If no one in the laboratory is analysis certified, they should refer to section
4.2.1.2 for submission instructions.
3.10 Analysis certified: An individual who is certified in BioNumerics gel analysis
3.11 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
3.13 “TIFF sent by CDC”: This is a part of the proficiency testing program where all laboratories analyze the
same TIFF sent to them by CDC
3.14 TIFF quality: The grading of the appearance and ease of analysis of a TIFF according to the PulseNet TIFF
Grading Guidelines (PNQ01). This is a main component of the evaluation of a TIFF submitted for
certification.
3.16 Proficiency testing evaluation: A report that contains the evaluation and results of the participant’s
proficiency test. See template in appendix PNQ04-3.
3.17 Proficiency testing packet: An electronic PDF file including a cover letter and the proficiency testing
evaluation that is emailed to the participant(s) who performed the PT, the primary PulseNet contact for the
laboratory and the participant’s laboratory director.
3.18 Certification file evaluator (evaluator): An individual who evaluates and signs off on the certification reports
submitted by PulseNet participants.
3.19 Certification file reviewer (reviewer): An individual who reviews and signs off on the certification reports
submitted by the certification file evaluator. There is a reviewer from the PulseNet Database Team and one
from the PulseNet Methods and Development Laboratory for each organism.
3.20 EDLB: Enteric Diseases Laboratory Branch
3.21 ITSO ticket: An internal CDC request submitted to CDC’s Information Technology Services Office via
http://intranet.cdc.gov/itso/ServiceDesk/default2.htm

4. RESPONSIBILITIES

4.1 Individuals performing PulseNet-related work (i.e., preparing PFGE gels and/or analyzing TIFF images) must
be certified before being able to participate in the proficiency testing program. See PNQ02 (SOP for
Certification of PulseNet Personnel) for information on becoming certified.
4.2 Certified individuals at PulseNet participating laboratories must:
VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

4.2.1 Perform proficiency testing annually for each pathogen in which they are certified, as part of continuing
in-house QA/QC. All certified individuals in a laboratory perform proficiency testing in-house, and one
set of results per pathogen per laboratory is selected and submitted to the organism specific proficiency
testing database at CDC.
4.2.1.1 Gel certified individuals run and image a gel containing the proficiency testing strain for each
pathogen in which they are certified. The TIFF generated by the participating laboratory (in-
house TIFF) is produced.
4.2.1.2 If the laboratory does not have an analysis certified individual on staff, the TIFF file generated
from the proficiency testing is sent via email to [email protected] with the subject line of
“organism Proficiency Testing.” In addition, the lab must provide lane information (indicate
what isolate is in what lane), including the standards.
4.2.1.3 Analysis certified individuals analyze the in-house TIFF and the TIFF sent by CDC in
BioNumerics for each pathogen in which they are certified. They upload the analyses and
TIFFs to the organism specific proficiency testing database at CDC.
4.2.1.4 An individual certified in gels and analysis may produce the TIFF generated by the
participating laboratory, analyze the proficiency testing TIFFs, and upload the results. For
some labs this may be a combined effort of multiple staff members, one certified to run gels
and one certified to perform analysis and upload results to CDC.

4.3 Individual(s) preparing isolates for shipment must:

4.3.1 Exchange emails with the evaluator(s) in order to decide which strains will be sent out.
4.3.2 Order and pick up shipment supplies and contact the CDC shipping department two months in advance to
schedule a shipping date and provide an estimate of what organisms and how many shipments will be
going out (Domestic and International). Note: You will need to request UN3733 shipping containers for
each shipment of E. coli O157:H7 and category B shipping bags for all other organisms at this time.
4.3.3 Prepare isolates for shipment as stated in the procedure section 5.2.

4.4 Individuals evaluating proficiency testing results (evaluators) must:

4.4.1 Send the individual(s) preparing isolates for shipment a list of certified laboratories scheduled to
participate, edit documentation for the proficiency testing round, select the TIFFs sent by CDC, assist in
selecting the proficiency testing strains, and contact the participating laboratories as stated under the
procedure section 5.1.
4.4.2 Confirm and update participant’s mailing addresses to create shipping labels
4.4.3 Submit an ITSO ticket to the attention of PulseNet’s IT Support (see contacts section 8) to notify them of
impending PT round. Give at least two weeks advance notice and make sure to note the expected shipment
week for cultures. Work with PulseNet IT Support to confirm participant’s access to the databases (go
through the list to make sure the contacts are still PulseNet participants and discuss any discrepancies in
records).
4.4.4 Assess the TIFF quality and ease of analysis of the TIFFs submitted for proficiency testing and assess the
gel analysis of proficiency testing submissions. All TIFFs and analyses submitted should be evaluated
based on the current PulseNet standards of TIFF quality (PNQ01) and gel analysis (PND04).
4.4.5 Use the “PT_Development” Microsoft Access database located in
\\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency Testing to evaluate the proficiency
testing submissions. A short cut to access the database may be saved on your desktop for easy access.
Instructions for using this database are listed under appendix PNQ04-6. Once all the reports are completed,
email the CDC reviewers to let them know to review the reports and insert electronic signature and date.
4.4.6 Modify reports based on CDC reviewers’ comments, if necessary. Resubmit reports to CDC reviewers as
soon as possible.
4.4.7 Create the cover letters using the template saved here
\\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency Testing\Templates
4.4.8 Convert final PT packets (cover letters and evaluations) into one PDF. Email the laboratory’s PT packet to
the participant(s), primary PulseNet contact and laboratory director.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

4.4.9 Using the existing templates \\cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency

Testing\Templates create summary documents once all results are complete and post within the PulseNet
Announcements forum on the PulseNet/OutbreakNet SharePoint site.

4.5 The Individuals reviewing proficiency testing evaluations submitted by the evaluator (reviewers) must:
4.5.1 Review the submitted evaluations and send any feedback to the evaluator (i.e. if you disagree with any of
the evaluator’s decisions or if you would like to edit any portion of the report).
4.5.2 The database team reviewer enters their scanned signature and date on each report in the PT Access
Database. Instructions are listed in appendix PNQ04-6. Once all the reports are signed, the database
reviewer should notify the laboratory reviewer that the reports are ready for approval.
4.5.3 The laboratory reviewer enters their scanned signature and date on each report in the PT Access Database.
Instructions are listed in appendix PNQ04-6. Once all the reports are signed, the laboratory reviewer should
notify the evaluator that the reports are ready for distribution.

5. PROCEDURE

5.1 Evaluators organizing an upcoming proficiency testing round:

5.1.1 Contact the individual(s) preparing isolates for shipment of the proficiency testing strains to select strains
for the round. Do this at least 6-8 weeks prior to desired shipment date.
5.1.2 Determine desired shipment date. Consult the individual(s) preparing isolates for shipment to make sure
the proposed date is acceptable. Fall and Spring rounds are typically ~6 months apart (i.e. Fall ships E. coli
O157:H7, Salmonella and Shigella in October, Spring ships Listeria, Campylobacter, Vibrio
parahaemolyticus and cholerae in April).
5.1.3 Edit the participants list, proficiency testing instructions, cover letters, and address labels for use in the
upcoming round of testing. Templates are located
\\cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency Testing\Templates
5.1.3.1 Email the participants list and address labels to the individual(s) preparing isolates for
shipment at least two weeks prior to shipment date
5.1.4 Email international participants to request any necessary import permits. Participants may email or fax
their permits to [email protected] or (404) 639-3333.
5.1.5 Determine which images (one per organism) to use for the “TIFF sent by CDC” portion of the test. If a
suitable TIFF cannot be found and exported from the national database then request one from the CDC
Methods and Development lab at least three weeks prior to shipment of PT strains. The TIFF needs to have
both enzymes present (with the exception of Campylobacter) and be of good to excellent quality.
5.1.6 Once the PT strains have shipped, email all the participating laboratories. Include the cover letter,
instructions, TIFFs sent by CDC and any other helpful information. Let them know when to expect the
strains to arrive. See appendix PNQ04-1 for a template.
5.1.6.1 Record which strains were used during this round in the “PT Strains_Tracking.doc” saved
\\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency Testing
5.1.6.2 Make sure that PulseNet IT Support emails the Proficiency Testing Database logins to all of
the participants the same week strains are shipped
5.1.7 Post the instructions and TIFFs sent by CDC on SharePoint within the PulseNet Announcements forum.
Title the posting “yyyy round Proficiency Testing.”
5.1.8 Email the participating laboratories approximately one to two weeks before the proficiency testing results
are due, giving notice of the impending deadline.
5.1.8.1 Promptly respond to any participants who have requested deadline extensions.

5.2 Individuals preparing and shipping strains for proficiency testing:

5.2.1 At 4-6 weeks before strains are shipped:
5.2.1.1 Decide which strains will be sent to certified labs for proficiency testing by selecting two strains
for each organism for preliminary testing.
5.2.1.2 Reconstitute at least two lyophilized vials of each strain, pick 2 different colonies from each
isolation plate and make PFGE plugs from the four cultures for each strain.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

5.2.2 Run a gel according to the appropriate organism specific PulseNet protocol with the new PFGE plugs.
Confirm the patterns by sending the TIFF to the evaluators to compare to the previous patterns obtained for
the strain.
5.2.3 At 3-4 weeks before strains are shipped:
5.2.3.1 Coordinate with the individual(s) organizing the proficiency testing round about the “TIFFs sent
by CDC.” If a suitable TIFF (excellent or good quality with H9812 standards in appropriate lanes
and an appropriate strain cut with the primary and secondary enzymes) cannot be found by the
evaluator, run a suitable TIFF that can be used for “the TIFF sent by CDC” portion of the PT.
5.2.3.2 Send the TIFF to the individuals organizing the proficiency testing to be sure that the gel image is
satisfactory and select strain to be used for each organism so there will be time to repeat it or find
another one, if necessary.
5.2.4 At 2-3 weeks before strains are shipped:
5.2.4.1 Receive list of participating laboratories organized by category of shipment. For example, E. coli
only, Salmonella only, Listeria only, E. coli and Salmonella, E. coli and Shigella, Salmonella and
Shigella, and E. coli, Salmonella, and Shigella.
5.2.4.2 Decide on the coding of the proficiency testing strains:
5.2.4.2.1 Strains are typically named using a two character descriptive of the organism (i.e.
Salmonella: SA), P for Proficiency, a two digit year (i.e. 14 for 2014), a dash (-), then the
CDC ID (i.e. 8099 for 80-99). For example the Salmonella PT strain with CDC ID 80-99
sent in 2013 was SAP13-8099. Consult with the evaluator for final strain designations.
5.2.4.2.2 Print out labels for:
5.2.4.2.2.1 Lyophilized vials – Include PulseNet Proficiency Test, Genus and
species, Lyophilization Date, PT strain ID (SAP13-8099), and
instructions for reconstituting (Reconstitute in 1ml H20).
5.2.4.2.2.2 Address/Return Labels for Category A shipments containing E. coli
O157:H7- Instructions should read (If not claimed after 5 days return
to: (Insert Shipper’s Name). U. S. Department of Health and Human
Services, Centers for Disease Control and Prevention,
Phone#:(XXX) XXX-XXXX, 1600 Clifton Rd, Mailstop X, Atlanta
GA, 30022” .
5.2.4.2.2.3 Labels defining contents of package- Ex. 1x1.0 ml Escherichia coli
O157:H7, 1 x 1.0 ml Salmonella enterica serotype Typhimurium.
5.2.4.2.3 Replace the original label on the vials with the proficiency testing lyophilized vial label
(this can be done the week before cultures are shipped).
5.2.4.2.4 Order and/or pick up enough of the following supplies for the shipments:
5.2.4.2.4.1 Shipping form CDC57.7, Rev. 7/2004 or submit electronic request (Scerison).
5.2.4.2.4.2 Peel-off mailing labels, CDC 0.689 Rev. 12/96 (Check with someone in the
shipping department because they can supply labels with sender, mailstop and
phone number already printed.)
5.2.4.2.4.3 Styrofoam sleeves- supplied by the Shipping Dept.
5.2.4.2.4.4 Pick up UN3733 shipping containers for each shipment of E. coli O157:H7 and
Category B shipping bags supplied by the shipping department for all other
organisms.
5.2.5 At 1-2 weeks before strains are shipped:
5.2.5.1 Coordinate with the shipping department to determine which days certain groups of shipments
will go out. For example, the week of PT shipping all shipments containing only E. coli O157
will go out Tuesday, all shipments containing E. coli O157, Salmonella and Shigella will go out
Wednesday in addition to the Salmonella only shipments. Adjust shipment date if necessary.
5.2.5.2 Send shipping department separate lists of addresses organized by category of shipment and
number each address. For Example, E. coli O157 only (and number 1-10), Salmonella and
Shigella (number 11-20), etc.
5.2.6 On the Thursday or Friday of the week before the strains are shipped:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

5.2.6.1 Separate lyophilized proficiency testing strains according to shipment type (1 culture, 2 cultures,
etc.) and put in labeled styrofoam sleeves (E and S; E, L and S, etc.). Refrigerate in shipment
specific containers according to category (UN3733 shipping containers for each shipment
containing E. coli O157:H7 and Category B shipping bags for all others)
5.2.6.2 Place a sticker indicating contents of shipment (refer to Section 5.2.4.2.2.3) on the outside of the
container (for E. coli O157:H7 place it on the canister, for all others place on Category B bag).
5.2.6.3 For domestic shipments, fill out information on Shipping Form (CDC57.7, Rev. 7/2004) or
submit electronic request through Scerison and address and phone number of recipient on mailing
labels. Separate into appropriate categories.
5.2.6.3.1 If the same items are being shipped to 3 or more domestic labs, only one shipping form has
to be filled out per order; in the “Ship To:” section, fill in the number of laboratories and
attach a separate sheet with the numbered list of names, addresses and phone numbers of
the labs receiving the cultures. The individual mailing labels still have to be completed for
each lab.
5.2.6.3.2 International shipments require a separate shipping form for each laboratory and
“Declaration for Export of Biologicals, Chemicals, Equipment, or Technical Data” Form
(CDC 50.117 Rev. 3/2000) in triplicate. Include a copy of an Import Permit for each
country, if required. Spell out both the genus and species name (i.e., Escherichia coli, and
not E. coli) on the forms. If shipping E. coli, Salmonella spp., Shigella spp., Vibrio spp.,
fill out an “Enteric Checklist Form”.
5.2.7 On the day of shipment
5.2.7.1 Bring necessary paperwork described above in section 5.2.6.3 and containers to the shipping
department by 10:00 am so they will have time to pack and ship the same day. Ask them to notify
you if shipments will be delayed.
5.2.7.2 Notify the evaluator once strains have shipped so they may notify laboratories and request that the
labs let you know if the package does not arrive by Friday, so it can be tracked. The labs do not
send the empty shipping boxes back to CDC.
5.3 Individuals at participating laboratories:
5.3.1 Must perform the proficiency testing according to the current proficiency testing instructions that are
emailed and posted on SharePoint within the PulseNet Announcements forum.
5.3.2 All certified individuals in a laboratory should perform proficiency testing annually as part of in-house
QA/QC. However, only one set of results per pathogen per laboratory should be chosen for
upload/submission to the organism specific PT database at CDC.
5.3.3 Laboratories must submit proficiency testing TIFFs and analyses performed by certified individuals.
Results submitted by non-certified individuals will automatically fail the proficiency testing round.
5.3.4 All results must be submitted correctly by the submission deadline to avoid penalty. Laboratories that
cannot meet the deadline may submit a request for extension to [email protected] before the submission
deadline.
5.3.5 Laboratories must submit a “submission email” as described in the PT instructions to the PFGE inbox after
their results have been submitted.
5.3.6 Laboratories must pass proficiency testing annually to maintain certification.
5.3.6.1 Laboratories that fail one proficiency testing round must resubmit the proficiency testing results
for that pathogen again using the same strains. The evaluator will email a failure notification letter
(see appendix PNQ04-5) to the participant, primary PulseNet contact, laboratory director and the
appropriate PulseNet Area Lab. Resubmission due dates will be determined by the evaluator.
5.3.6.2 Laboratories that fail for a particular organism two times in a row will lose their certification and
must submit routine gels to [email protected] . Individuals at those laboratories must submit new
certification files and pass certification again before reinstatement of their certification. Please see
PNQ02 for PulseNet Certification information.
5.3.6.3 CDC has the right to revoke certifications at any time

5.4 Individuals evaluating proficiency testing results (evaluators):

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

5.4.1 In the last week of the deadline for submissions, send an email reminder to all the participating laboratories
and check the submissions.
5.4.2 Open the PT_Development Access Database to begin evaluations
5.4.3 Evaluate one pathogen at a time
5.4.3.1 Click the green plus sign next to “New Analysis (PFGE)” to open the evaluation template. Fill in
the appropriate information in the “Create New Analysis” box that opens (Year, Season, LabID,
Organism and Resubmission). Click OK.
5.4.3.2 There are four tabs across the top: Gel Prep, Gel Analysis, Submission of Results and Other Info
5.4.3.2.1 Type in any comments or notes in the “Comments” column next to the appropriate section
(e.g., normalization, band marking, etc.). The comments should explain any point
deductions and/or any PFGE patterns that did not match the majority of submissions
received.
5.4.3.2.2 In the “PT Comments” section, type in any TIFF grading comments that would explain
the reason for the grade and add any more suggestions for TIFF improvement, any more
detailed notes about the band marking or band resolution, etc.
5.4.3.2.3 Use the submission email sent by each laboratory to fill in the Other Info tab fields (e.g.,
person who prepared gel, equipment used, etc.).
5.4.3.2.4 Enter your scanned signature and date the reports as instructed in appendix PNQ04-6
5.4.3.2.5 Enter the images into each report as instructed in appendix PNQ04-6
5.4.3.2.6 Save the reports under
\\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency Testing\yyyy
round\Reports\organism and notify the reviewer once they are ready for their review
5.4.4 TIFF evaluation
5.4.4.1 In the organism specific PT Admin database objectively grade the in-house TIFFs according to
the PulseNet TIFF Grading Guidelines. TIFFs should receive a grade of excellent, good, fair, or
poor. Record the corresponding points and any comments for improvement in the proficiency
testing report (Gel Prep tab in Access).Refer to the Standard Comments for Troubleshooting for
example statements \\cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Troubleshooting.
5.4.5 Gel analysis evaluation
5.4.5.1 In the organism specific PT Admin database pull up all the proficiency testing submissions for
that round in BioNumerics. Check to make sure all submissions are there, that they are linked
properly, and that the strain numbers are entered correctly into the “Key field.”
5.4.5.2 Divide all submissions for each pathogen into the two proficiency testing parts, the “TIFF sent by
CDC” and the “TIFF generated in-house.” Create a dendrogram for each enzyme of all the
submissions for each part of the proficiency testing. Printing the dendrograms to write comments
and note discrepancies as a whole may be helpful while evaluating and when you begin to
compile the summary documents to post on SharePoint. Saving the submissions as comparisons
may also be helpful.
5.4.5.3 Record the corresponding points and any comments for improvement in the proficiency testing
report (Gel Analysis tab in Access).
5.4.5.4 “TIFF sent by CDC” evaluation
5.4.5.4.1 Compare band markings to the CDC submission, which should be analyzed according to
the PulseNet Gel Analysis Guidelines.
5.4.5.4.2 On the dendrogram, pay attention to any areas with discrepancies as compared to the CDC
submission and the majority of submissions. Record any comments about the discrepancy
and at what molecular weight the discrepancy took place on the appropriate laboratory’s
evaluations. Only deduct points for areas that are marked differently than they are
resolved.
5.4.5.4.3 Open the TIFFs and check for any normalization discrepancies. This could be indicated by
bands on a submission that are consistently lower or higher than other submissions
received. For the “TIFF sent by CDC,” all laboratories are analyzing the same TIFF, so all
bands should be the same down the dendrogram. Record any comments for normalization
or if the submission should be failed because of a normalization error.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

5.4.5.4.4 Record any submissions that are to have band marking points deducted, and record the
number of deducted points.
5.4.5.4.4.1 Three points are deducted for each discrepancy ≥300 kb (eyeball the top part of
the lane)
5.4.5.4.4.2 Two points are deducted for each discrepancy from 299-100 kb (eyeball the
middle of the lane)
5.4.5.4.4.3 One point is deducted for each discrepancy <100 kb (eyeball the bottom part of
the lane)
5.4.5.5 “TIFF generated in-house” evaluation
5.4.5.5.1 Look at the band markings compared to the CDC submission which should be analyzed
according to the PulseNet Gel Analysis Guidelines (PND04). This will help determine the
quality of the band resolution of the submissions.
5.4.5.5.2 Compare each submission (i.e., each lane) to the actual TIFF submitted by the
participating laboratory.
5.4.5.5.2.1 Note whether the band marking matches the TIFF, according to the PulseNet
Gel Analysis Guidelines (PND04). If the band marking does not visually match
the TIFF, make a comment on the appropriate laboratory’s evaluation about the
discrepancies and at what molecular weight the discrepancies took place. If the
band marking matches the TIFF but is different from the majority of
submissions received, note that on the dendrogram as well. If necessary, deduct
appropriate number of points as described in section 5.4.5.3.4.
5.4.5.5.2.2 Check for any normalization discrepancies on the TIFF. Normalization issues
could also be seen by looking at the dendrogram for bands on a submission that
are consistently lower or higher than other submissions received. Record any
comments for normalization or if the submission should be failed because of a
normalization error.
5.4.5.5.2.3 Toggle between the normalization step and analysis step to be sure that there
are no bands marked above or below the bands of the reference system (when
automatic band marking is used sometimes bands are marked outside of the
reference system)
5.4.6 Cover Letter
5.4.6.1 Use the cover letter template in appendix PNQ04-4 and edit the document as necessary. Save on
the network drive as “LabID_Cover Letter.doc”
\\cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency Testing\yyyy
Round\Reports\Cover Letters. Do this for each participating laboratory.
5.4.7 When all reports are finalized, create a PT summary posting showing summary statistics for the proficiency
testing round (e.g., number of laboratories participating, number that failed, number of excellent TIFFs,
etc.). Templates can be found \\cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency
Testing\Templates.
5.4.8 Create summary documents for posting on SharePoint. These documents should summarize the equipment
and enzyme manufacturers used for the proficiency testing survey and also show pertinent information
about the proficiency testing results. For examples please see the following: “TIFF examples.ppt” and
“Summary of Enzyme and Equipment.xls” Templates can be found
\\cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency Testing\Templates.

5.5 Individuals reviewing proficiency testing packets (reviewers).

5.5.1 The review will consist of checking the TIFF quality assessment and the comments associated with each
analysis discrepancy. The reviewer will enter their electronic signature if he/she agrees with the evaluator’s
assessment of the proficiency testing files. If the reviewer does not agree with the evaluator’s assessment,
the reviewer must contact the evaluator and discuss modifying the proficiency testing report.

5.6 Evaluators sending out proficiency testing packets:

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

5.6.1 Convert final PT packets (cover letters and evaluations) into one PDF. Save the final PDFs on the network
drive \\cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency Testing\yyyy round\Final
Reports. Email the laboratory’s PT packet (final PDF) to the participant(s), main PulseNet contact and
laboratory director. Refer back to section 5.3.6 if a laboratory should fail the proficiency test.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

6. FLOW CHART:

Individual is certified in gels, analysis, or both gels and analysis.

• In order to maintain certification, PT must be performed for each
organism in which an individual is certified

CDC mails PT strains to laboratories with at least one

certified person
• Fall round: E. coli, Salmonella, and Shigella
• Spring round: Campylobacter, Listeria, and Vibrio

Each person performs PT

• Laboratory chooses one set of
results to submit per organism

Run “In-house TIFF”*

Analyze “In house TIFF” and

upload results to appropriate PT
database

Analyze “TIFF sent by CDC” and

upload results to appropriate PT
database

Submissions evaluated, reports

emailed to participants, and overall
results posted on SharePoint

VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

7. BIBLIOGRAPHY:

8. CONTACTS:
8.1 CDC PulseNet Database Unit
(404) 639-4558
[email protected]
8.2 CDC PulseNet Methods and Development Laboratory Unit
(404) 639-4558
[email protected]
8.3 CDC Shipping: Yvonne Stifel
(404) 639-3355
[email protected]
8.4 PulseNet Information Technology
Brenda Brown
(404) 639-3942
[email protected]

9. AMENDMENTS:
9.1 July 2014: Entire document was updated to reflect a paperless process for sending proficiency testing
instructions and final evaluations. PT instructions and results will no longer be mailed; all instructional
documents will be emailed and posted on SharePoint within the PulseNet Announcements forum. PT
database logins will be emailed separately. All final evaluations will be emailed to the participant(s), the
primary PulseNet contact and the laboratory director.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

Appendix PNQ04-1

Template email announcing PT shipment of strains and TIFFs sent by CDC

Greetings everyone,

It is time again for PulseNet Proficiency Testing (PT). Persons who were certified for [Insert the organisms
included in the round of PT] on or before [month date, year] are expected to complete PT this round. Each
participating laboratory receives a PT strain and a “TIFF sent by CDC” for the organism(s) for which the laboratory
is currently certified. Please make sure to share this information with other certified personnel within your
laboratory. The strains and emails are only being sent to one person per laboratory, however all instructions and
TIFFs have also been posted on SharePoint within the PulseNet Announcements forum.

The PT strains for [Insert the organisms included in the round of PT] were shipped on [date]. PT database logins
are being sent in a separate email this week. If you do not receive the PT strains and/or PT database logins by [date],
please let us know.

The attached TIFFs are to be analyzed and submitted for the “TIFFs sent by CDC” portion of PT. Lane information
may be found in the attached instructions.

If you have any questions, email [email protected] with “Proficiency Testing” in the subject line.

Thank you,

[Name of contact(s)]
PulseNet QA/QC Program
[Phone number(s)]

VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

Appendix PNQ04-3

Proficiency Testing Evaluation Template

NOTE: When evaluating a PT resubmission, the title of the report should be (Round #, i.e. 10th) PulseNet
Proficiency Testing Survey (round yyyy) Resubmission Report Form

(Round #, i.e. 10th) PulseNet Proficiency Testing Survey (round yyyy) Report Form

Organism: Date lyophilized: Date results received

TIFF by CDC:
Laboratory: Date strains shipped: TIFF by lab:

No. of Points
Possible Points Comments
Received
I. Gel Preparation (50 pts)
Excellent (Pass 20 pts)
A. Overall gel and TIFF quality Good (Pass 15 pts)
Fair (Pass 10 pts)
Poor (Fail)
B. Correct pattern (visual) attained in:
1. Lane containing [primary enzyme] pattern Pass (13 pts) or Fail
2. Lane containing [secondary enzyme] pattern Pass (13 pts) or Fail

C. Were directions followed?

1. Correct file name 1 pt
2. Run in requested lanes 1 pt
3. Run on routine gel 1 pt
4. Correct standard and pattern Pass (1 pt) or Fail

II. Gel Analysis - TIFF generated by participating lab (22 pts)

A. Correct normalization for:

1. Lane containing [primary enzyme] pattern 6 pts or Fail
2. Lane containing [secondary enzyme] pattern 6 pts or Fail

B. Band marking compared to Guidelines for:

1. Lane containing [primary enzyme] pattern 5 pts
2. Lane containing [secondary enzyme] pattern 5 pts

III. Gel Analysis - TIFF sent by CDC (22 pts)

A. Correct normalization for:

1. Lane containing [primary enzyme] pattern 6 pts or Fail
2. Lane containing [secondary enzyme] pattern 6 pts or Fail
VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

B. Band marking compared to Guidelines for:

1. Lane containing [primary enzyme] pattern 5 pts
2. Lane containing [secondary enzyme] pattern 5 pts

IV. Submission of Results (6 pts)

A. All results sent/uploaded for:

1. TIFF generated by participating laboratory Pass (1 pt) or Fail
2. TIFF sent by CDC Pass (1 pt) or Fail
B. TIFF file generated in-house was uploaded 2 pts
C. Correct strain numbers used in BioNumerics 1 pt
D. Lanes linked correctly in BioNumerics 1 pt

Total points received

Overall proficiency testing result Pass or Fail

Person who prepared the gel:

Person who performed the analysis:
Person who uploaded the analysis:
Equipment used:
Enzymes ordered from:

Proficiency Testing Comments:

Some or all items in I-A, I-B, I-C, II-A, III-A, and IV-A on the report form are in Pass / Fail format. If you fail any one of the
questions, you fail the proficiency testing round. Laboratories that pass will accumulate the specified points. For band marking, all
isolates were compared to the PulseNet Gel Analysis Guidelines. If band marking differed from the guidelines, up to 5 points were
deducted for each band difference according to the position of the difference in three organism-dependent zones as follows: -3 points
in the top 1/3 of the gel; -2 points in the middle 1/3 of the gel; -1 point each (up to 4 points) in the bottom 1/3 of the gel. A passing
score is >=85% (>=85/100).

Proficiency Testing Analysis:

Performed By: ________________________________________________________ Date of Report:

name, [email protected], phone number
VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

Reviewed By: ________________________________________________________ Date of Review:

name, [email protected], phone number

Approved By: ________________________________________________________ Date of Approval:

name, [email protected], phone number

[A COPY OF THE SUBMITTED IMAGE SHOULD BE INSERTED HERE]

Appendix PNQ04-4

Proficiency Testing Cover Letter Template

Month yyyy

Dear Participant(s), Primary PulseNet Contact, and Lab Director,

The Fall/Spring yyyy round of PulseNet Proficiency Testing for Organisms has been completed. The results for your
laboratory are enclosed.

Below is the breakdown of the number of laboratories who have passed the Fall/Spring yyyy round:

Salmonella: 81/84 laboratories passed* 6 are currently pending

E. coli: 78/80 laboratories passed* 4 are currently pending
Shigella: 61/62 laboratories passed* 5 are currently pending

*Laboratories that did not pass were notified in month along with a request for resubmission.

A score of 85 points or higher was needed to pass this proficiency testing round. A thorough description of the
scoring system is included in each report, as well as specific comments and suggestions for your laboratory, when
applicable. Detailed summary reports and tips for improvement are posted in PulseNet Announcements on
SharePoint. PulseNet laboratory staff should have access to this forum.
VERSION: REPLACED BY: AUTHORIZED BY:

Page 14 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

Successful completion of the Proficiency Testing for the specified organisms maintains your standing as a certified
PulseNet laboratory. If you have any questions please contact me or send an email to [email protected]. We
appreciate your participation and continued support of PulseNet.

Sincerely,

name
contact info

Cc: Food Safety Team, Association of Public Health Laboratories

PulseNet Unit, Centers for Disease Control and Prevention

Appendix PNQ04-5

Proficiency Testing Notification of Failure Template

[Save on CDC Letterhead]

[Date]

[Name and info of lab director],

The [laboratory] PulseNet laboratory did not pass the [round yyyy] round of the PulseNet Proficiency Testing for
[organism]. [Insert reason for fail]. The [laboratory] laboratory must resubmit the [error example: analysis of the
“TIFF sent by CDC” with proper normalization] by [due date.]

[Person] was notified on [date] and a resubmission was requested by the above mentioned deadline. Comments and
suggestions for improvement were provided. PulseNet participants are always reminded to contact their designated
PulseNet Area Laboratory or CDC with any questions or for troubleshooting assistance.

If no resubmission is received, or if the resubmission is not satisfactory, this round of proficiency testing is
considered failed. If the resubmitted results are satisfactory, the round is recorded as a pass. Resubmission
evaluations will be emailed to participants and their laboratory directors upon completion.

Please let us know if you have any questions or concerns.

Thank you,

Name
Contact info

VERSION: REPLACED BY: AUTHORIZED BY:

Page 15 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

Appendix PNQ04-6

Instructions for Setup and Use of the PT_Development Access Database

The location of the database is \\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\QAQC\Proficiency Testing

For those who evaluate, review or approve PT results you may wish to create a desktop shortcut for easy access.

1. To add your personal information (name, phone, email and signature), open the PT_Development Access
Database and click the View/Edit Roles button

2. Click the plus sign next to your role (evaluators click “perform”, database reviewers click “review”,
laboratory reviewers click “approve”). Then Type your first and last name into the next available box.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 16 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

3. Click the plus sign next to your name to enter the remaining information. Click “Pull from Contacts table”
to auto populate your email and phone number (the information is pulled from the PulseNet Contacts
Database). Then click “Browse” to import your electronic signature.
Note 1: You must have already saved a scanned image of your signature on a private drive (such as your
desktop or C drive). The image must be cropped so that your signature does not appear shrunken (zoom in
to crop). The default image width will be 120 and height will be 30.
Note 2: It is highly recommended that you test using your signature in a single report before you use it to
mass review.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 17 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

4. Instructions for Evaluator:

To begin the evaluation, click the green plus sign next to “New Analysis”

The year will auto populate (make sure it is for the correct round of PT). Use the pull-down menus to
populate the rest of the data fields.

There are four tabs across the top of the page: Gel Prep, Gel Analysis, Submission of Results and Other
Info. Click the tab to enter information into the report form as you are evaluating the PT submission. Enter
comments and points under the appropriate headings.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 18 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

When you get to the Other Info tab, use the information provided in the PT submission emails sent from the
participating laboratories to complete this section (strain and received dates, person who…, and other). For
the Comments, enter any general comments you have for improving gel quality, analysis or submission.

When you are ready to score the overall results, click the auto-total button to auto populate the total points
and final result (pass/fail) section. If you want to go ahead and generate the final report then, use the pull-
down to select your name and click the “generate report” button. This will generate a single excel file. If
you wish to wait until you are finished with all the reports to sign off, then use the tool in the main screen
to mass sign.

5. Instructions for Mass Review/Approve: First enter the year (i.e. 2014), then click “Browse” to navigate
to the location where all of the reports are saved (the evaluator will notify the reviewers of the
location).Click the pull-down arrow next to Reviewer to select your name from the list. Then click the
magnifying glass button next to “Review All Reports in Folder”. This should automatically insert your
signature and the current date into all of the reports. Please check a few to make sure this worked properly.
Note: The arrows point to the fields used by the Database Reviewers. The laboratory reviewer should use
the section of tools under “Approve All Reports in Folder” in the same order as described above and
pictured below to complete the review/approval process.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 19 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ04
Effective Date:
THE PULSENET PFGE PROFICIENCY TESTING PROGRAM 07 01 14

4
2
1 3

6. If any files weren’t able to be reviewed or approved, you’ll get a pop-up message telling you which ones
had an error. Otherwise, you’ll receive a confirmation message that says “All files successfully
reviewed/approved”.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 20 of 20
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

1. PURPOSE: To describe the procedure for certifying PulseNet personnel to enable full
participation in PulseNet MLVA activities.

2. SCOPE: This procedure applies to all PulseNet personnel performing MLVA and creating peak
files.

3. DEFINITIONS/TERMS:
MLVA: Multiple-locus variable-number tandem repeat analysis
DNA: Deoxyribonucleic acid
PCR: Polymerase chain reaction
CDC: Centers for Disease Control and Prevention
SOP: Standard Operating Procedure

4. RESPONSIBILITIES:

4.1 Individuals performing PulseNet MLVA-related work must submit certification file(s) and have
them reviewed before being able to submit peak files to the PulseNet MLVA Database.
4.1.1 Submitted certification files must document the submitter’s highest level of competence in
producing peak files.
4.1.2 Individuals can be certified for peak files only at this time.

5. PROCEDURE:

5.1 PulseNet participants request the E. coli O157 and S. enterica serotypes Typhimurium and
Enteritidis MLVA certification sets from CDC ([email protected]) if they do not already have them.
5.2 CDC sends the requested certification set and detailed instructions (see Appendices PNQ05-1
through PNQ05-5) to reconstitute the cultures and primers, make DNA templates, perform PCR
and fragment analysis and export the peak file from the sequencer in the “.CSV” format according
to the standardized laboratory protocol (PNL19, PNL21, and PNL27).
5.3 Peak file(s) are submitted to CDC for review. See Appendices PNQ05-2, PNQ05-3 and PNQ05-4
for submission instructions.
5.4 Submitters are notified in writing of the results of their certification file evaluation.
5.4.1 If the submitted certification file passes the certification evaluation, the submitter is considered
certified as long as they remain in their current laboratory and that laboratory successfully
completes annual proficiency testing. If a person relocates to a different PulseNet laboratory,
they must be recertified.
5.4.2 If the submitted certification files do not pass the certification evaluation
5.4.2.1 The individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved.
5.4.2.2 If the submitter fails certification three times, the individual will not be allowed to
submit again for six months. Before resubmitting, the individual will be expected to work
with CDC until satisfactory results are achieved. This includes, but is not limited to
troubleshooting and training in the PulseNet MLVA protocol.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:

8.1 Eija Hyytia-Trees, D.V.M., Ph.D.

PulseNet Next Generation Subtyping Methods Unit, EDLB, DFWED, CDC
(404) 639-3672
[email protected]

8.2 Patti Lafon, M.S.

PulseNet Next Generation Subtyping Methods Unit, EDLB, DFWED, CDC
(404) 639-2828
[email protected]

9. AMENDMENTS:

9.1 Appendix PNQ05-3 Instructions: PulseNet Certification for MLVA Peak Files of S. Typhimurium -
Beckman Coulter CEQ 8000 Platform added 2/5/2008.

9.2 Appendix PNQ05-3: The internal ladder isolates were assigned CDC identification numbers
10/6/2009.

9.3 Appendix PNQ05-4: Primer reconstitution instructions were added 3/2/2010. A list of certification
package contents was added to the top of each PNQ05-2 and PNQ05-3.

9.4 Appendix PNQ05-4 was added 3/26/2010. This document details the certification procedure for the
S. enterica serotype Enteritidis MLVA. Former Appendix PNQ05-4 (primer reconstitution
instructions) was renamed PNQ05-5 and was amended to reflect the addition of S. Enteritidis
MLVA certification.

9.5 Appendix PNQ05-1: Removed section regarding TSA Stabs: Day 1. All certification strains and
controls are now lyophilized 4/19/2013.

9.6 Document title now includes the 8800 and GeXP Genetic Analyzers 4/19/2013..

9.7Appendix PNQ05-2: Revised contents to include 11 lyophilized vials. Updated Eija Trees’ job title
and affiliation. Included the GeXP and 8800 Genetic Analyzers in the text 4/19/13 .
9.8Appendix PNQ05-3: Revised contents to include 11 lyophilized vials. Updated Eija Trees’ job title
and affiliation. Included the GeXP and 8800 Genetic Analyzers in the text 4/19/13 .

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

9.7Appendix PNQ05-4: Revised contents to include 11 lyophilized vials. Updated Eija Trees’ job title
and affiliation. Included the GeXP and 8800 Genetic Analyzers in the text 4/19/13.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

Appendix PNQ05-1

Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) E. coli O157:H7,S.

Typhimurium, and S. enterica serotype Enteritidis

Biological Safety Warning: E. coli 0157:H7 and Salmonella strains are considered Level 2 biological
agents by the U.S. Department of Health and Human Services. Use appropriate precautions when
handling the vial or culture. Carry out laboratory work in a biological safety cabinet when applicable to
ensure aseptic conditions and personal safety.

Note: Store the lyophilized cultures and TSA stabs at 4°C in the dark until they are reconstituted and
subcultured.

Materials Needed:
Sterile sturdy forceps
1 ml pipetman
1 ml sterile pipet tips
1 µl sterile inoculating loop

Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water or Trypticase Soy Broth (TSB)
70% isopropyl alcohol

Procedure for Reviving Cultures:

Lyophilized cultures: Day 1

1. Document the isolate number (s) and the date(s) lyophilized for your records. Wipe the aluminum
cover and outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the
aluminum cover and rubber stopper from the vial containing the lyophilized culture. Wipe the outside
of the rubber stopper and neck of the vial with isopropyl alcohol before removing the stopper.

2. Re-suspend the lyophilized cells with 1.0 ml of sterile grade reagent water. Allow to stand for a few
minutes and/or mix gently to produce a uniform suspension. With an inoculating loop, streak a small
amount of this suspension onto a blood agar plate (BAP) and incubate at 37°C overnight.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

Days 2 and 3

1. Check the BAP; if the culture appears pure, pick an isolated colony, and streak it on a fresh BAP;
incubate at 37°C overnight. Use the growth from this plate to make DNA templates of the certification
strains. Transfer culture to fresh medium and incubate at 37°C overnight; this will ensure that the same
culture can be retested, if necessary.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

Appendix PNQ05-2

Instructions: PulseNet Certification for MLVA Peak Files of E. coli O157:H7-Beckman Coulter
CEQ 8000/8800/GeXP Platform

Dear PulseNet Participant,

This package should contain:

- 16 vials of primers (please see Appendix PNQ05-5 for primer reconstitution instructions)
- 11 vials of lyophilized cultures of E. coli O157:H7:
o 8 Certification Strains
o 1 Positive Control Strain (EDL933)
o 2 Internal Ladder Strains (EC04PN0139 and EC04PN0570)

After the certification strains have been reconstituted according to the directions in Appendix PNQ05-
1, streak each culture onto agar plates (overnight incubation at 37oC), pick an isolated colony, and
subculture to another plate. Use the growth from the second plate to make the DNA templates. Please
let me know if this package does not arrive in a satisfactory condition, or if the cultures are not viable.
Please, make a stock culture (freeze at -70°C) of each of the strains according to your
laboratory’s policy within 1 week from receiving them. Long-term storage of these cultures will
ensure the availability of the PulseNet certification set for future use, including MLVA
certification of additional personnel.

The strain numbers of the E. coli cultures are as follows:

CDC# 01-98 CDC# 05-98 CDC# 07-98 CDC# 08-98 CDC# 12-98 CDC# 24-98

CD# 11 (G5286) CDC# 48 (G7602)

Please follow these supplemental instructions for testing the certification isolates by MLVA.
Refer to the “Laboratory standard operating procedure for PulseNet MLVA of Shiga toxin-
producing Escherichia coli O157 (STEC O157)-Beckman Coulter CEQ 8000/8800/GeXP
Platform (PNL19)” for detailed instructions.

1. Make DNA templates from each test isolate, positive control EDL933, and internal ladder
isolates EC04PN0139 and EC04PN0570.

2. Perform PCR and fragment analysis following the instructions of the standard protocol with
the possible exception of the primer concentration modifications your laboratory may have had
to make to optimize the PCR assays.

a. Make sure to include your PulseNet laboratory ID (the unique identifier code that was
assigned to your laboratory by CDC PulseNet) in front of the CDC strain ID number
VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

and your initials after the strain ID number, i.e. follow the strain ID format
labID_CDC01-98xx.
3. Export the peak file from the sequencer in the .CSV format. Please notice that in addition to
the certification set isolates, the peak file must also contain the positive control strain EDL933
in duplicate for each reaction, one negative control for each reaction, the internal ladder in
duplicate, and the molecular size standard peaks (D1 peaks).

a. Name the peak files according to the standardized PulseNet naming system:

Use the laboratory ID that was assigned to your laboratory by CDC PulseNet for the
first two to four letters of the file. The next 2 spaces will indicate the year the file was
generated. The next 4 spaces indicate the month and the date the run was performed.
For example: GA090219.csv is a peak file run on Feb 19th, 2009 at the GA Public
Health Laboratory. If multiple runs are performed on a same day, differentiate the
peak files using sequential numbers, for example GA090219-1, GA090219-2.

4. Send the peak file to CDC PulseNet at [email protected] within four weeks after receiving the
strains.

a. In the email to CDC, include E. coli O157 MLVA Certification in the subject line.

Currently, for E. coli O157, an individual may be certified for peak file submission only. Once the E.
coli O157 national MLVA database is available on-line, individuals may also be certified for analysis.

After the peak files are submitted, the PulseNet certification file evaluator will analyze the files and
inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of
receiving the files. If the peak file is satisfactory, the person who submitted the file will be eligible to
send peak files to PulseNet for analysis. If the submitted certification files are not satisfactory, the
individual will need to review the troubleshooting comments received from the evaluator and resubmit
once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work
with CDC until satisfactory results are achieved.

Please let me know if you have questions or further clarification is needed.

Good luck,

Eija Trees, D.V.M., Ph.D.

Unit Chief
PulseNet Next Generation Subtyping Methods Unit
EDLB, DFWED, CDC
Tel: 404-639-3672
E-mail: [email protected]

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

Appendix PNQ05-3

Instructions: PulseNet Certification for MLVA Peak Files of S. enterica serotype Typhimurium -
Beckman Coulter CEQ 8000/8800/GeXP Platform

Dear PulseNet Participant,

This package should contain:

- 14 vials of primers (please see Appendix PNQ05-5 for primer reconstitution instructions)
- 11 vials of lyophilized cultures of Salmonella Typhimurium
o 8 Certification strains
o 1 Positive control (LT2)
o 2 Internal Ladder isolates (2009K0825 and 2009K0826)

After the strains have been reconstituted according to the directions in Appendix PNQ05-1, streak each
culture onto agar plates (overnight incubation at 37oC), pick an isolated colony, and subculture to
another plate. Use the growth from the second plate to make the DNA templates. Please let me know if
this package does not arrive in a satisfactory condition, or if the cultures are not viable. Please, make a
stock culture (freeze at -70°C) of each of the strains according to your laboratory’s policy within
1 week from receiving them. Long-term storage of these cultures will ensure the availability of
the PulseNet certification set for future use, including MLVA certification of additional
personnel.

The strain numbers of the S.enterica serotype Typhimurium cultures are as follows:

CDC# 61-99 CDC# 63-99 CDC# 76-99 CDC# 78-99 CDC# 80-99 CDC# 81-99

CD# 83-99 CDC# H8290

Please follow these supplemental instructions for testing the certification isolates by MLVA.
Refer to the “Laboratory standard operating procedure for PulseNet MLVA of Salmonella
enterica serotype Typhimurium (S. Typhimurium) - Beckman Coulter CEQ 8000 Platform
(PNL21)” for detailed instructions.

1. Make DNA templates from each test isolate, positive control LT2, and internal ladder
isolates 2009K0825 and 2009K0826.

2. Perform PCR and fragment analysis following the instructions of the standard protocol with
the possible exception of the primer concentration modifications your laboratory may have had
to make to optimize the PCR assays.

a. Make sure to include your PulseNet laboratory ID (the unique identifier code that was
assigned to your laboratory by CDC PulseNet) in front of the CDC strain ID number
VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

and your initials after the strain ID number, i.e. follow the strain ID format
labID_CDC61-99xx.

3. Export the peak file from the sequencer in the .CSV format. Please notice that in addition to
the certification set isolates, the peak file must also contain the positive control strain LT2 in
duplicate for each reaction, one negative control for each reaction, the internal ladder in
duplicate, and the molecular size standard peaks (D1 peaks).

a. Name the peak files according to the standardized PulseNet naming system:

Use the laboratory ID that was assigned to your laboratory by CDC PulseNet for the
first two to four letters of the file. The next 2 spaces will indicate the year the file was
generated. The next 4 spaces indicate the month and the date the run was performed.
For example: GA090219.csv is a peak file run on Feb 19th, 2009 at the GA Public
Health Laboratory. If multiple runs are performed on a same day, differentiate the
peak files using sequential numbers, for example GA090219-1, GA090219-2.

4. Send the peak file to CDC PulseNet at [email protected] within four weeks after receiving the
strains.

a. In the email to CDC, include S. Typhimurium MLVA Certification in the subject

line.

Currently, for S. Typhimurium, an individual may be certified for peak file submission only. Once the
S. Typhimurium national MLVA database is available on-line, individuals may also be certified for
analysis.
After the peak files are submitted, the PulseNet certification file evaluator will analyze the files and
inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of
receiving the files. If the peak file is satisfactory, the person who submitted the file will be eligible to
send peak files to PulseNet for analysis. If the submitted certification files are not satisfactory, the
individual will need to review the troubleshooting comments received from the evaluator and resubmit
once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work
with CDC until satisfactory results are achieved.
Please let me know if you have questions or further clarification is needed.
Good luck,
Eija Trees, D.V.M., Ph.D.
Unit Chief PulseNet Next Generation Subtyping Methods Unit
EDLB, DFWED, CDC
Tel: 404-639-3672
E-mail: [email protected]
VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

Appendix PNQ05-4

Instructions: PulseNet Certification for MLVA Peak Files of S. enterica serotype Enteritidis -
Beckman Coulter CEQ 8000/8800/GeXP Platform

Dear PulseNet Participant,

This package should contain:

- 14 vials of primers (please see Appendix PNQ05-5 for primer reconstitution instructions)
- 11 vials of lyophilized cultures of Salmonella Enteritidis
o 8 Certification Strains
o 1 Positive control (K1891)
o 2 Internal Ladder isolates (H9560 and 2010K0017)

According to the directions in Appendix PNQ06-1, streak each culture onto agar plates (overnight
incubation at 37oC), pick an isolated colony, and subculture to another plate. Use the growth from the
second plate to make the DNA templates. Please let me know if this package does not arrive in
satisfactory condition, or if the cultures are not viable. Please, make a stock culture (freeze at -70°C)
of each of the strains according to your laboratory’s policy within 1 week from receiving them.
Long-term storage of these cultures will ensure the availability of the PulseNet certification set
for future use, including MLVA certification of additional personnel.

The strain numbers of the S. enterica serotype Enteritidis cultures are as follows:

CDC# K2148 CDC# H9654 CDC# 2009K0432 CDC# K3307 CDC# J0932

CDC# K4417 CDC# K2158 CDC# K0746

Please follow these supplemental instructions for testing the certification isolates by MLVA.
Refer to the “Laboratory standard operating procedure for PulseNet MLVA of Salmonella
enterica serotype Enteritidis- Beckman Coulter CEQ 8000/8800/GeXP Platform (PNL27)” for
detailed instructions.

1. Make DNA templates from each test isolate, positive control K1891, and internal ladder
isolates H9560 and 2010K0017.

2. Perform PCR and fragment analysis following the instructions of the standard protocol with
the possible exception of the primer concentration modifications your laboratory may have had
to make to optimize the PCR assays.

a. Make sure to include your PulseNet laboratory ID (the unique identifier code that
was assigned to your laboratory by CDC PulseNet) in front of the CDC strain ID

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

number and your initials after the strain ID number, i.e. follow the strain ID format
labID_CDCK2148xx.
3. Export the peak file from the sequencer in the .CSV format. Please notice that in addition to
the certification set isolates, the peak file must also contain the positive control strain K1891 in
duplicate for each reaction, one negative control for each reaction, the internal ladder in
duplicate, and the molecular size standard peaks (D1 peaks).

a. Name the peak files according to the standardized PulseNet naming system:

Use the laboratory ID that was assigned to your laboratory by CDC PulseNet for
the first two to four letters of the file. The next 2 spaces will indicate the year the
file was generated. The next 4 spaces indicate the month and the date the run was
performed. For example: GA090219.csv is a peak file run on Feb 19th, 2009 at the
GA Public Health Laboratory. If you perform multiple runs on a same day,
differentiate the peak files using sequential numbers, for example GA090219-1,
GA090219-2.

4. Send the peak file to CDC PulseNet at [email protected] within four weeks after receiving the
strains.

a. In the email to CDC, include S. Enteritidis MLVA Certification in the subject line.

Currently, for S. enterica serotype Enteritidis, an individual may be certified for peak file submission
only. Once the national S. Enteritidis MLVA database is available on-line, individuals may also be
certified for analysis.

After the peak files are submitted, the PulseNet certification file evaluator will analyze the files and
inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of
receiving the files. If the peak file is satisfactory, the person who submitted the file will be eligible to
send peak files to PulseNet for analysis. If the submitted certification files are not satisfactory, the
individual will need to review the troubleshooting comments received from the evaluator and resubmit
once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work
with CDC until satisfactory results are achieved.
Please let me know if you have questions or further clarification is needed.
Good luck,
Eija Trees, D.V.M., Ph.D.
Unit Chief PulseNet Next Generation Subtyping Methods Unit
EDLB, DFWED, CDC
Tel: 404-639-3672
E-mail: [email protected]
VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

Appendix PNQ05-5

Instructions: Reconstitution of Primers for E. coli O157:H7 and Salmonella enterica serotypes
Typhimurium and Enteritidis

E. coli O157:H7:

Reconstitution of the primers:

The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock, please
reconstitute the primers by adding following amounts of distilled water:

Amount of primer in the vial Water needed for a 100 µM stock

10 nM 100 µl
40 nM 400 µl

Preparation of the working concentrations from the 100 µM stock:

Working concentration: Water (µl) + primer (µl)

25 µM 30.0 + 10.0
5 µM 47.5 + 2.5
2.5 µM 48.75 + 1.25
1 µM 99.0 + 1.0

Salmonella Typhimurium:

Reconstitution of the primers:

The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock, please
reconstitute the primers by adding following amounts of distilled water:

Amount of primer in the vial Water needed for a 100 µM stock

10 nM 100 µl
20 nM 200 µl
40 nM 400 µl

Preparation of the working concentrations from the 100 µM stock:

Working concentration: Water (µl) + primer (µl)

5 µM 47.5 + 2.5
2.5 µM 48.75 + 1.25

VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ05
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN Effective Date:
COULTER CEQ 8000/8800/GeXP PLATFORM 04 13 13

Salmonella Enteritidis:

Reconstitution of the primers:

The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock, please
reconstitute the primers by adding following amounts of distilled water:

Amount of primer in the vial Water needed for a 100 µM stock

10 nM 100 µl
20 nM 200 µl

Preparation of the working concentrations from the 100 µM stock:

Working concentration: Water (µl) + primer (µl)

12.5 µM 43.75 + 6.25
2.5 µM 48.75 + 1.25
1 µM 99.0 + 1.0

VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

1. PURPOSE: To describe the procedure for certifying PulseNet personnel to enable full
participation in PulseNet MLVA activities.

2. SCOPE: This procedure applies to all PulseNet personnel performing MLVA and creating
peak files.

3. DEFINITIONS/TERMS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 PCR: Polymerase chain reaction
3.3 DNA: Deoxyribonucleic acid
3.4 CDC: Centers for Disease Control and Prevention
3.5 SOP: Standard Operating Procedure

4. RESPONSIBILITIES:

4.1 Individuals performing PulseNet MLVA-related work must submit certification file(s) and
have them reviewed before being able to submit peak files to the PulseNet MLVA Database.
4.1.1 Submitted certification files must document the submitter’s highest level of competence
in producing peak files.
4.1.2 Individuals can be certified for peak files only at this time.

5. PROCEDURE:

5.1 PulseNet participants request the E. coli O157 and Salmonella enterica serotypes
Typhimurium and Enteritidis MLVA certification sets from CDC ([email protected]) if they do
not already have them.
5.2 CDC sends the requested certification set and detailed instructions (see Appendices PNQ06-1
through PNQ06-5) to reconstitute the cultures and primers, make DNA templates, perform
PCR and fragment analysis and export the peak file from the sequencer in the “.txt” format
according to the standardized laboratory protocols (PNL23, PNL24, PNL26, PNL28, PNL29,
PNL30).
5.3 Peak file(s) are submitted to CDC for review. See Appendices PNQ06-2, PNQ06-3 and
PNQ06-4 for submission instructions.
5.4 Submitters are notified in writing of the results of their certification file evaluation.
5.4.1 If the submitted certification file passes the certification evaluation, the submitter is
considered certified as long as they remain in their current laboratory and that laboratory
successfully completes annual proficiency testing. If a person relocates to a different
PulseNet laboratory, they must be recertified.
5.4.2 If the submitted certification files do not pass the certification evaluation
5.4.2.1 The individual will need to review the troubleshooting comments received from
the evaluator and resubmit once results have improved.
5.4.2.2 If the submitter fails certification three times, the individual will not be allowed to
submit again for six months. Before resubmitting, the individual will be expected to

VERSION: REPLACED BY: AUTHORIZED BY:

Page 1 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

work with CDC until satisfactory results are achieved. This includes, but is not
limited to troubleshooting and training in the PulseNet MLVA protocol.

6. FLOW CHART:

7. BIBLIOGRAPHY:

8. CONTACTS:

8.1 Eija Hyytia-Trees, D.V.M., Ph.D.

PulseNet Next Generation Subtyping Methods Unit, EDLB, DFWED, CDC
(404) 639-3672
[email protected]

8.2 Patti Lafon, M.S.

PulseNet Next Generation Subtyping Methods Unit, EDLB, DFWED, CDC
(404) 639-2828
[email protected]

9. AMENDMENTS:

9.1 Appendix PNQ06-3 was added 10/1/2009. This document details the certification procedure
for the S. enterica serotype Typhimurium MLVA.

9.2 Appendix PNQ06-4: Primer reconstitution instructions were added 3/2/2010. A list of
certification package contents was added to the top of each PNQ06-2 and PNQ06-3.

9.3 Appendix PNQ06-4 was added 3/17/2010. This document details the certification procedure
for the S. enterica serotype Enteritidis MLVA. Former Appendix PNQ06-4 (primer
reconstitution instructions) was renamed PNQ06-5 and was amended to reflect the addition of S.
Enteritidis MLVA certification.

9.4 Document Title now includes the 3500 Genetic Analyzer 4/19/2013.

9.5 Appendix PNQ06-1: Removed section regarding TSA Stabs: Day 1. All certification strains
and controls are now lyophilized 4/19/2013.

9.6 Appendix PNQ06-2: Revised contents to include 11 lyophilized vials. Updated Eija Trees’
job title and affiliation. Included the reference to PNL28 (3500) in the text 4/19/13 .

9.7 Appendix PNQ06-3: Revised contents to include 11 lyophilized vials. Updated Eija Trees’
job title and affiliation. Included the reference to PNL29 (3500) in the text 4/19/13 .

VERSION: REPLACED BY: AUTHORIZED BY:

Page 2 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

9.8 Appendix PNQ06-4: Revised contents to include 11 lyophilized vials. Updated Eija Trees’
job title and affiliation. Included the reference to PNL30 (3500) in the text 4/19/13 .

VERSION: REPLACED BY: AUTHORIZED BY:

Page 3 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

Appendix PNQ06-1

Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) E. coli O157:H7, S.

enterica serotype Typhimurium and S. enterica serotype Enteritidis

Biological Safety Warning: E. coli 0157:H7, S. enterica serotypes Typhimurium and Enteritidis
strains are considered Level 2 biological agents by the U.S. Department of Health and Human
Services. Use appropriate precautions when handling the vial or culture. Carry out laboratory
work in a biological safety cabinet when applicable to ensure aseptic conditions and personal
safety.

Note: Store the lyophilized cultures and TSA stabs at 4°C in the dark until they are reconstituted
and subcultured.

Materials Needed:
Sterile sturdy forceps
1 ml pipetman
1 ml sterile pipet tips
1 µl sterile inoculating loop

Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water or Trypticase Soy Broth (TSB)
70% isopropyl alcohol

Procedure for Reviving Cultures:

Lyophilized cultures: Day 1

1. Document the isolate number (s) and the date(s) lyophilized for your records. Wipe the
aluminum cover and outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically
remove the aluminum cover and rubber stopper from the vial containing the lyophilized culture.
Wipe the outside of the rubber stopper and neck of the vial with isopropyl alcohol before
removing the stopper.

2. Re-suspend the lyophilized cells with 1.0 ml of sterile grade reagent water. Allow to stand
for a few minutes and/or mix gently to produce a uniform suspension. With an inoculating loop,
streak a small amount of this suspension onto a blood agar plate (BAP) and incubate at 37°C
overnight.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 4 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

Days 2 and 3

1. Check the BAP; if the culture appears pure, pick an isolated colony, and streak it on a fresh
BAP; incubate at 37°C overnight. Use the growth from this plate to make DNA templates of the
certification strains. Transfer culture to fresh medium and incubate at 37°C overnight; this will
ensure that the same culture can be retested, if necessary.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 5 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

Appendix PNQ06-2

Instructions: PulseNet Certification for MLVA Peak Files of E. coli O157:H7-Applied

Biosystems Genetic Analyzer 3130/3500 Platform

Dear PulseNet Participant,

This package should contain:

- 16 vials of primers (please see Appendix PNQ06-5 for primer reconstitution instructions)
- 11 vials of lyophilized cultures of E. coli O157:H7
o 8 Certification Strains
o 1 Positive Control Strain (EDL933)
o 2 Internal Ladder Strains (EC04PN0139 and EC04PN0570)

After the strains have been reconstituted according to the directions in Appendix PNQ06-1,
streak each culture onto agar plates (overnight incubation at 37oC), pick an isolated colony, and
subculture to another plate. Use the growth from the second plate to make the DNA templates.
Please let me know if this package does not arrive in satisfactory condition, or if the cultures are
not viable. Please, make a stock culture (freeze at -70°C) of each of the strains according to
your laboratory’s policy within 1 week from receiving them. Long-term storage of these
cultures will ensure the availability of the PulseNet certification set for future use,
including MLVA certification of additional personnel.

The strain numbers of the E. coli cultures are as follows:

CDC# 01-98 CDC# 05-98 CDC# 07-98 CDC# 08-98 CDC# 12-98 CDC# 24-98

CD# 11 (G5286) CDC# 48 (G7602)

Please follow these supplemental instructions for testing the certification isolates by
MLVA. Refer to PNL23 (3130) or PNL28 (3500) for detailed instructions.

1. Make DNA templates from each test isolate, positive control EDL933, and internal
ladder isolates EC04PN0139 and EC04PN0570.

2. Perform PCR and fragment analysis following the instructions of the standard protocol
with the possible exception of the primer concentration modifications your laboratory
may have had to make to optimize the PCR assays.

a. Make sure to include your PulseNet laboratory ID (the unique identifier code
that was assigned to your laboratory by CDC PulseNet) in front of the CDC strain
ID number and your initials after the strain ID number, i.e. follow the strain ID
format labID_CDC01-98xx.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 6 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

3. Export the peak file from the sequencer in the .txt (tab-delimited) format. Please notice
that in addition to the certification set isolates, the peak file must also contain the positive
control strain EDL933 in duplicate for each reaction, one negative control for each
reaction, the internal ladder in duplicate, and the molecular size standard peaks (R peaks).

a. Name the peak files according to the standardized PulseNet naming system:

Use the laboratory ID that was assigned to your laboratory by CDC

PulseNet for the first two to four letters of the file. The next 2 spaces will
indicate the year the file was generated. The next 4 spaces indicate the
month and the date the run was performed. For example: GA090219.txt is a
peak file run on Feb 19th, 2009 at the GA Public Health Laboratory. If you
perform multiple runs on a same day, differentiate the peak files using
sequential numbers, for example GA090219-1, GA090219-2

4. Send the peak file to CDC PulseNet at [email protected] within four weeks after receiving
the strains.

a. In the email to CDC, include E. coli O157 MLVA Certification in the subject line.

Currently, for E. coli O157, an individual may be certified for peak file submission only. Once
the E. coli O157 national MLVA database is available on-line, individuals may also be certified
for analysis.

After the peak files are submitted, the PulseNet certification file evaluator will analyze the files
and inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four
weeks of receiving the files. If the peak file is satisfactory, the person who submitted the file will
be eligible to send peak files to PulseNet for analysis. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved. If the submitter fails certification three
times, the individual will not be allowed to submit again for six months. Before resubmitting, the
individual will be expected to work with CDC until satisfactory results are achieved.

Please let me know if you have questions or further clarification is needed.

Good luck,

Eija Trees, D.V.M., Ph.D.

Unit Chief
PulseNet Next Generation Subtyping Methods Unit
EDLB, DFWED, CDC
Tel: 404-639-3672
E-mail: [email protected]

VERSION: REPLACED BY: AUTHORIZED BY:

Page 7 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

Appendix PNQ06-3

Instructions: PulseNet Certification for MLVA Peak Files of S. enterica serotype

Typhimurium-Applied Biosystems Genetic Analyzer 3130/3500 Platform

Dear PulseNet Participant,

This package should contain:

- 14 vials of primers (please see Appendix PNQ06-5 for primer reconstitution instructions)
- 11 vials of lyophilized cultures of Salmonella Typhimurium
o 8 Certification Strains
o 1 Positive Control Strain (LT2)
o 2 Internal Ladder Strains (2009K0825 and 2009K0826)

After the strains have been reconstituted according to the directions in Appendix PNQ06-1,
streak each culture onto agar plates (overnight incubation at 37oC), pick an isolated colony, and
subculture to another plate. Use the growth from the second plate to make the DNA templates.
Please let me know if this package does not arrive in satisfactory condition, or if the cultures are
not viable. Please, make a stock culture (freeze at -70°C) of each of the strains according to
your laboratory’s policy within 1 week from receiving them. Long-term storage of these
cultures will ensure the availability of the PulseNet certification set for future use,
including MLVA certification of additional personnel.

The strain numbers of the S. enterica serotype Typhimurium cultures are as follows:

CDC# 61-99 CDC# 63-99 CDC# 76-99 CDC# 78-99 CDC# 80-99 CDC# 81-99

CDC# 83-99 CDC# H8290

Please follow these supplemental instructions for testing the certification isolates by
MLVA. Refer to PNL24 (3130) or PNL29 (3500) for detailed instructions.

1. Make DNA templates from each test isolate, positive control LT2, and internal ladder
isolates 2009K0825 and 2009K0826.

2. Perform PCR and fragment analysis following the instructions of the standard protocol
with the possible exception of the primer concentration modifications your laboratory
may have had to make to optimize the PCR assays.

a. Make sure to include your PulseNet laboratory ID (the unique identifier code
that was assigned to your laboratory by CDC PulseNet) in front of the CDC strain
ID number and your initials after the strain ID number, i.e. follow the strain ID
format labID_CDC61-99xx.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 8 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

3. Export the peak file from the sequencer in the .txt (tab-delimited) format. Please notice
that in addition to the certification set isolates, the peak file must also contain the positive
control strain LT2 in duplicate for each reaction, one negative control for each reaction,
the internal ladder in duplicate, and the molecular size standard peaks (R peaks).

a. Name the peak files according to the standardized PulseNet naming system:

Use the laboratory ID that was assigned to your laboratory by CDC

PulseNet for the first two to four letters of the file. The next 2 spaces will
indicate the year the file was generated. The next 4 spaces indicate the
month and the date the run was performed. For example: GA090219.txt is a
peak file run on Feb 19th, 2009 at the GA Public Health Laboratory. If you
perform multiple runs on a same day, differentiate the peak files using
sequential numbers, for example GA090219-1, GA090219-2.

4. Send the peak file to CDC PulseNet at [email protected] within four weeks after receiving
the strains.

a. In the email to CDC, include S. Typhimurium MLVA Certification in the subject

line.

Currently, for S. enterica serotype Typhimurium, an individual may be certified for peak file
submission only. Once the national S. Typhimurium MLVA database is available on-line,
individuals may also be certified for analysis.

After the peak files are submitted, the PulseNet certification file evaluator will analyze the files
and inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four
weeks of receiving the files. If the peak file is satisfactory, the person who submitted the file will
be eligible to send peak files to PulseNet for analysis. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved. If the submitter fails certification three
times, the individual will not be allowed to submit again for six months. Before resubmitting, the
individual will be expected to work with CDC until satisfactory results are achieved.

Please let me know if you have questions or further clarification is needed.

Good luck,

Eija Trees, D.V.M., Ph.D.

Unit Chief
PulseNet Next Generation Subtyping Methods Unit
EDLB, DFWED, CDC
Tel: 404-639-3672
E-mail: [email protected]
VERSION: REPLACED BY: AUTHORIZED BY:

Page 9 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

Appendix PNQ06-4
Instructions: PulseNet Certification for MLVA Peak Files of S. enterica serotype
Enteritidis - Applied Biosystems Genetic Analyzer 3130/3500 Platform

Dear PulseNet Participant,

This package should contain:

- 14 vials of primers (please see Appendix PNQ06-5 for primer reconstitution instructions)
- 11 vials of lyophilized cultures of Salmonella Enteritidis
o 8 Certification Strains
o 1 Positive Control Strain (K1891)
o 2 Internal Ladder Strains (H9560 and 2010K0017)

According to the directions in Appendix PNQ06-1, streak each culture onto agar plates
(overnight incubation at 37oC), pick an isolated colony, and subculture to another plate. Use the
growth from the second plate to make the DNA templates. Please let me know if this package
does not arrive in satisfactory condition, or if the cultures are not viable. Please, make a stock
culture (freeze at -70°C) of each of the strains according to your laboratory’s policy within
1 week from receiving them. Long-term storage of these cultures will ensure the availability
of the PulseNet certification set for future use, including MLVA certification of additional
personnel.

The strain numbers of the S. enterica serotype Enteritidis cultures are as follows:

CDC# K2148 CDC# H9654 CDC# 2009K0432 CDC# K3307 CDC# J0932

CDC# K4417 CDC# K2158 CDC# K0746

Please follow these supplemental instructions for testing the certification isolates by
MLVA. Refer to PNL26 (3130) or PNL30 (3500) for detailed instructions.

1. Make DNA templates from each test isolate, positive control K1891, and internal
ladder isolates H9560 and 2010K0017.

2. Perform PCR and fragment analysis following the instructions of the standard protocol
with the possible exception of the primer concentration modifications your laboratory
may have had to make to optimize the PCR assays.

a. Make sure to include your PulseNet laboratory ID (the unique identifier code
that was assigned to your laboratory by CDC PulseNet) in front of the CDC strain
ID number and your initials after the strain ID number, i.e. follow the strain ID
format labID_CDCK2148xx.

VERSION: REPLACED BY: AUTHORIZED BY:

Page 10 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

3. Export the peak file from the sequencer in the .txt (tab-delimited) format. Please notice
that in addition to the certification set isolates, the peak file must also contain the positive
control strain K1891 in duplicate for each reaction, one negative control for each
reaction, the internal ladder in duplicate, and the molecular size standard peaks (R peaks).

a. Name the peak files according to the standardized PulseNet naming system:

Use the laboratory ID that was assigned to your laboratory by CDC

PulseNet for the first two to four letters of the file. The next 2 spaces will
indicate the year the file was generated. The next 4 spaces indicate the
month and the date the run was performed. For example: GA090219.txt is a
peak file run on Feb 19th, 2009 at the GA Public Health Laboratory. If you
perform multiple runs on a same day, differentiate the peak files using
sequential numbers, for example GA090219-1, GA090219-2.

4. Send the peak file to CDC PulseNet at [email protected] within four weeks after receiving
the strains.

a. In the email to CDC, include S. Enteritidis MLVA Certification in the subject line.

Currently, for S. enterica serotype Enteritidis, an individual may be certified for peak file
submission only. Once the national S. Enteritidis MLVA database is available on-line,
individuals may also be certified for analysis.

After the peak files are submitted, the PulseNet certification file evaluator will analyze the files
and inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four
weeks of receiving the files. If the peak file is satisfactory, the person who submitted the file will
be eligible to send peak files to PulseNet for analysis. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved. If the submitter fails certification three
times, the individual will not be allowed to submit again for six months. Before resubmitting, the
individual will be expected to work with CDC until satisfactory results are achieved.

Please let me know if you have questions or further clarification is needed.

Good luck,

Eija Trees, D.V.M., Ph.D.

Unit Chief
PulseNet Next Generation Subtyping Methods Unit
EDLB, DFWED, CDC
Tel: 404-639-3672
E-mail: [email protected]

VERSION: REPLACED BY: AUTHORIZED BY:

Page 11 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

Appendix PNQ06-5

Instructions: Reconstitution of Primers for E. coli O157:H7 and Salmonella enterica

serotypes Typhimurium and Enteritidis

E. coli O157:H7:

Reconstitution of the primers:

The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock,
please reconstitute the primers by adding following amounts of distilled water:

Amount of primer in the vial Water needed for a 100 µM stock

10 nM 100 µl
40 nM 400 µl

Preparation of the working concentrations from the 100 µM stock:

Working concentration: Water (µl) + primer (µl)

25 µM 30.0 + 10.0
5 µM 47.5 + 2.5
2.5 µM 48.75 + 1.25
1 µM 99.0 + 1.0

Salmonella Typhimurium:

Reconstitution of the primers:

The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock,
please reconstitute the primers by adding following amounts of distilled water:

Amount of primer in the vial Water needed for a 100 µM stock

10 nM 100 µl
20 nM 200 µl
40 nM 400 µl

Preparation of the working concentrations from the 100 µM stock:

Working concentration: Water (µl) + primer (µl)

25 µM 30.0 + 10.0
5 µM 47.5 + 2.5
2.5 µM 48.75 + 1.25

VERSION: REPLACED BY: AUTHORIZED BY:

Page 12 of 13
 STANDARD OPERATING PROCEDURE FOR CODE: PNQ06
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED Effective Date:
BIOSYSTEMS GENETIC ANALYZER 3130/3500 PLATFORM 04 19 13

Salmonella Enteritidis:

Reconstitution of the primers:

The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock,
please reconstitute the primers by adding following amounts of distilled water:

Amount of primer in the vial Water needed for a 100 µM stock

10 nM 100 µl
20 nM 200 µl

Preparation of the working concentrations from the 100 µM stock:

Working concentration: Water (µl) + primer (µl)

12.5 µM 43.75 + 6.25
2.5 µM 48.75 + 1.25

VERSION: REPLACED BY: AUTHORIZED BY:

Page 13 of 13
READING CONTROL SHEET FOR:
STANDARD OPERATING PROCEDURES FOR THE PULSENET QA/QC
PROGRAM (PNQ)

NAME DATE COMMENTS SIGNATURE

By signing above, you are indicating that you have read and understood all SOPs
included in the PNQ section of this manual.