Molecular methods in

Diagnostic Microbiology
Dr. Shaheda Anwar
MBBS MPhil PhD
Associate professor
Department of microbiology & immunology
BSMMU
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Polymerase chain reaction (PCR)

• The ability to amplify a specific DNA sequence
• Target amplification: involves making many copies of a specific DNA sequence.

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• PCR is the first and prototypical method for amplifying target nucleic acid

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DNA structure

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Polymerase Chain Reaction (PCR)

• Kary Mullis visualized the idea of

amplifying DNA in vitro in 1983
while driving one night on a
5
California Highway.

Kary Mullis is legendary for his invention of

PCR, which redefined the world of DNA,
genetics, and forensic science. He is also a
surfer, a veteran of Berkeley in the sixties,
and perhaps the only Nobel laureate to
describe a possible encounter with aliens.

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1986 prototype PCR machine (thermocycler)

“This machine is called Baby Blue”

Light Cycler PCR machine on display at the

Science Museum, London
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This is a very, very old PCR machine. The photo’s description says: “Early model of a
Polymerase Chain Reaction machine (“Gene Machine”), featuring three constant
D R .temperature
SH@HED@ ANW@R baths and a robotic action that moves samples between the baths”.
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How does a PCR machine look like today?

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Nucleic acid extraction

• The purpose is to release the nucleic acid from the cell
• The target nucleic acid should be free of contamination with protein,
carbohydrate, lipids, or other nucleic acid, that is, DNA free of RNA or RNA free of
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DNA.
• The initial release of the cellular material is achieved by breaking the cell and
nuclear membranes (cell lysis).
• Lysis must take place in conditions that will not damage the nucleic acid.
• Following lysis, the target material is purified, and then the concentration and
purity of the sample can be determined.

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Preparing the Sample

• Nucleic acid is routinely isolated from human, fungal, bacterial, and viral sources in the
clinical laboratory
• The initial steps in nucleic acid isolation depend on the nature of the starting material. 10

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Nucleated Cells in Suspension (Blood and

Bone Marrow Aspirates)
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• Depending on the type of specimen sent for
analysis, pretreatment may be necessary to make
cells available from which the nucleic acid will be
extracted 12

• white blood cells (WBCs) in blood or bone marrow

specimens are removed from red blood cells (RBCs)
and other blood components by either differential
density gradient centrifugation or differential lysis.
• For differential density gradient centrifugation,
whole blood or bone marrow mixed with isotonic
saline is overlaid with Ficoll.

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Tissue samples
• Fresh or frozen tissue samples must be dissociated before DNA isolation procedures can be
started.
• Grinding the frozen tissue in liquid nitrogen, homogenizing the tissue, or simply mincing
the tissue using a scalpel can disrupt whole tissue samples. 13

• Fixed, embedded tissue may be deparaffinized by soaking in xylene (a mixture of three

isomers of dimethylbenzene)
• After xylene treatment, the tissue is usually rehydrated by soaking it in decreasing
concentrations of ethanol.

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Microorganisms
• Several enzyme products (e.g., lysozyme or zymolyase) that digest cell wall polymers
are commercially available.
• Alternatively, cell walls can be broken mechanically by grinding or by vigorously mixing
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with glass beads
• Treatment with detergent (1% sodium dodecyl sulfate) and strong base (0.2 M NaOH) in
the presence of Tris base, EDTA and glucose can also break bacterial cell walls.
• Boiling in dilute sucrose, Triton X-100 detergent, Tris buffer, and EDTA after lysozyme
treatment releases DNA that can be immediately precipitated with alcohol

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Organic isolation method

• After release of DNA from the cell, further purification requires removal of contaminating
proteins, lipids, carbohydrates, and cell debris.
• Using a combination of high salt, low pH, and an organic mixture of phenol and chloroform.
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• The combination readily dissolves hydrophobic contaminants, such as lipids and
lipoproteins , collects cell debris, and strips away most DNA-associated proteins
• To avoid RNA contamination, RNAse, an enzyme that degrades RNA, can be added

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Inorganic isolation method

• Inorganic DNA extraction is sometimes called “salting out ”.
• It makes use of low pH and high salt conditions to selectively precipitate proteins,
leaving the DNA in solution.
• The DNA can then be precipitated using isopropanol, pelleted and resuspended in TE
buffer or water. 17

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Solid-Phase Isolation
• More rapid and comparably effective DNA extraction can be performed
using solid matrices to bind and wash the DNA.
• Silica-based products were shown to effectively bind DNA in high salt
conditions
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• Columns come in various sizes, depending on the amount of DNA to be

isolated. Columns used in the clinical laboratory are most often small “spin
columns” that fit inside microcentrifuge tubes.

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For solid-phase separation, the cell lysate is applied to a column in high salt buffer, and the
DNA in solution adsorbs to the solid matrix. Carrier RNA or DNA is applied with the DNA to
enhance recovery, preventing irreversible binding of the small amount of target DNA. The
carrier must be nucleic acid of more than 200 nucleotides in length to bind to the silica
membrane.

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DNA absorbed to magnetic beads is washed by suspension of the beads in buffer and
collection of the beads using a magnet applied to the outside of the tube while the
buffer is aspirated or poured off. The DNA IQ system (Promega) uses a magnetic resin
that holds a specific amount of DNA (100 ng). When the DNA is eluted in 100 μL, the
DNA concentration is known (1 ng/μL) and ready for analysis.

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Extraction with Chelating Resin

• Chelex is a cation-chelating resin that can be used for simple extraction of DNA

• A suspension of 10% chelex resin beads is mixed with specimen, and the cells are 22
lysed by boiling. After centrifugation of the suspension,
• DNA in the supernatant is cooled and may be further extracted with chloroform before
use in amplification procedures.
• This method is most commonly used in forensic applications but may also be useful for
purification of DNA from clinical samples and fixed, paraffin embedded specimens

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Isolation of RNA
• It is necessary to allocate a separate RNAse-free (RNF) area of the laboratory for storage
of materials and specimen handling intended for RNA analysis.
• Gloves must always be worn in the RNF area.
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• Disposables (tubes, tips, etc.) that come in contact with the RNA should be kept at this
location and never be touched by ungloved hands.
• Articles designated DNAse-free/RNF by suppliers may be used directly from the package.
• Although reusable glassware is seldom used for RNA work, it can be rendered RNF.
• After cleaning, glassware must be baked for 4 to 6 hours at 400¡C to inactivate the
RNAses.

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Cell lysis step

• The cell lysis step for RNA isolation is performed in detergent or phenol in the
presence of high salt (0.2 to 0.5 M NaCl) or RNAse inhibitors.
• Guanidine isothiocyanate (GITC) is a strong denaturant of RNAses and can be used
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instead of high salt buffers.
• Strong reducing agents such as 2-mercaptoethanol may also be added during this
step.

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• Once the cells are lysed, proteins can be extracted by organic means
• Acid phenol: chloroform: isoamyl alcohol (25:24:1) solution efficiently extracts RNA.
• Chloroform enhances the extraction of the nucleic acid by denaturing proteins and
promoting phase separation.
• Isoamyl alcohol prevents foaming.
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• For RNA, the organic phase must be acidic (pH 4–5). The acidity of the organic phase is
adjusted by overlaying it with buffer of the appropriate pH.
• In some isolation procedures, DNAse is added at the lysis step to eliminate
contaminating DNA.
• Alternatively, RNAse-free DNAse also may be added directly to the isolated RNA at
the end of the procedure.

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• After phase separation, the upper aqueous phase containing the RNA is
removed to a clean tube, and the RNA is precipitated by addition of two
volumes of ethanol or one volume of isopropanol.

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Components of PCR
• DNA template
• May derived from patients genomic or mitochondrial DNA or from viruses, bacteria, fungi
or parasite that might be infectious to the patients
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• Genomic DNA will have only 1 or 2 copies per cell equivalent of single copy genes to serve
as amplification targets
• For routine analysis, 100 ng to 1ug of DNA is usually used
• Best template- free of contaminating proteins, and without nicks or breaks that can stop
DNA synthesis or cause misincorporation of nucleotide bases.
• High GC content and secondary structure – difficult to optimize the amplificaiton.

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Primers
• Critical component- determines the specificity of the PCR
• Chemically manufactured on a DNA synthesizer.
• Primer sequences are submitted in text form, along with other specifications- amount,
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target, degree of purification, and any modifications such as biotin or fluorescent dyes
• Ss DNA fragments, usually 20 to 30 bases long. They are designed to contain the specific
sequence complementary to sites flanking the region to be analysed

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• Forward primer: hybridizes to the complimentary strand just 5’ to the sequence to be

amplified and reverse primer hybridizes to the complimentary strand just 3’ to the
sequence to be amplified
• The placement of the primers will also dictate the size of the amplified product

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Deoxyribonucleotide bases
• An equimolar mixture of the 4 deoxynucleotide triphosphate (dNTPs) – adenine (A),
guanine (G), Thymine (T), cytosine ( c) – is added to the synthesis reaction in concentrations
sufficient to support the exponential increase of copies of the template.
• Standard procedure requires 0.1 to 0.5 mM concentration of each nucleotide 30

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DNA polymerase
• Isolated from the thermophilic bacterium Thermus aquaticus
• DNA polymerase can be added at the beginning of the procedure and would
maintain its activity throughout the heating and cooling cycles
• Taq polymerase: T comes from the genus Thermus, aq comes from acquaticus 31

• Stoffel segment: Modified version of Taq polymerase, lack 289 N-terminal

amino acids of Taq polymerase and its inherent 3’ to 5’ exonuclease activity. Half
life is 2 times more, MgCl2 concentration range is broader ( 2 to 10mM).
Recommended for Allele specific PCR
• ThermoSequenase and T7 sequenace: For Sanger Sequencing

Despite its thermostabiliy, It is subjected to loss of activity in adverse situation-

vigorous agitation- mechanical shearing and alternating of the secondary and tertiary
structures and irreversible denaturation
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Steps of PCR
• Denaturation: The first step of a PCR where the sample is heated to separate or denature the two
strands of the DNA (>90∘C)
• Annealing: Following the denaturation step, the reaction temperature is lowered (usually 3-50C below
the Tm of primer) to allow the oligonucleotide primers to bind to the single strands of the template 32

DNA.
• Extension: The final step of the PCR where the temperature is raised, typically to 72¡C, allowing specific
enzymes to synthesize a new DNA strand complementary to the DNA template.

Step Temperature (∘C) Time (sec)

Denaturation 90-96 20-60
Annealing 50-70 20-90
Extension 68-75 10-60
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Step 1: Denaturation

• As in DNA replication, the two strands in the DNA double helix need to be separated.
• The separation happens by raising the temperature of the mixture, causing the hydrogen
bonds between the complementary DNA strands to break. This process is called denaturation.
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• This is accomplished by heating the sample at 94C to 96C for several seconds to several
minutes, depending on the template

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Step 2: Annealing
• Primers bind to the target DNA sequences and initiate polymerization. This can only occur once
the temperature of the solution has been lowered. One primer binds to each strand.
• 2 oligonucleotide primers that will prime the synthesis of DNA anneal ( hybridizes) to
complementary sequences on the template
• Primer determines the specificity of the amplification. 36

• Annealing temp be optimized with the primers and reaction conditions.

• A starting point can be determined using the Tm of the primer sequence

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Step 2: Annealing

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Step 3: Extension
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• A DNA polymerase enzyme joins free DNA nucleotides together

• The order in which the free nucleotides are added is determined by the sequence of
nucleotides in the original (template) DNA strand.
• The result of one cycle of PCR is two double-stranded sequences of target DNA, each
containing one newly made strand and one original strand.
• The cycle is repeated many times (usually 20–30) as most processes using PCR need large
quantities of DNA. It only takes 2–3 hours to get a billion or so copies. 38

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• The nucleotide sequence orders of forward and reverse primers are designed using genomic
sequences available from National Center for Biological Information or other resources
• Primers can be designed manually or alternatively primer sequences can be automatically 39
generated by software programs from an input target sequence and chosen parameters , such as
desired amplification length.
• Melting temp, or Tm of the primers, length, GC content %- important
• Tm of the primers- starting point for setting the optimal annealing temp
• Both forward and reverse primer should have same Tm , so both will hybridize optimally at
the same annealing temp.

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At the end of the 3 steps or one cycle, one copy of double

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stranded DNA has been replicated into 2 double stranded
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• The products of first cycle are again

replicated in the 2nd PCR cycle,
yielding four copies of the target
region. 41

• At each cycle , the number of copies

of the target region doubles

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Post-PCR analysis/processing
• Post PCR detection system must accurately and reproducibly
reflect the nature and quantity of the starting DNA template.
• The simplest method uses agarose gel electrophoresis.
• After the electrophoresis, PCR products can be visualized by
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staining the gel with fluorescent dye such as ethidium
bromide which binds to DNA and intercalates between the
stacked bases.
• Confirmation of size of the DNA product is done by
comparing the size with DNA ladder. The appearance of
discrete band of the correct size may be indicative of a
successful PCR amplification.

 Detection of PCR products by gel electrophoresis. The first lane (left) contains molecular weight markers (fragments o
known size). The next six lanes are PCR products, followed by a reagent blank control for contamination (lane 8). (From
DR. SH@HED@ ANW@R Buckingham L. Molecular Diagnostics. 2nd ed. Philadelphia, PA:F.A. Davis; 2011, with permission.)
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Other methods used for post PCR analysis are

• (1) Sequencing of the PCR product
• the gold standard but expensive and not widely available.
• PCR product may be sequenced directly or cloned before sequencing.
• it is the test of choice in outbreak situations where there are serious public health and/or medical-legal
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implications.
• Sequencing can be used to confirm results of other molecular epidemiological assays. As a matter of
fact, all other assays can be considered as simpler screening assays.
• (2) Restriction Fragment Length Polymorphism (RFLP) - very simple, rapid and economical technique
but the result may be difficult to read.
• (3) Hybridization with a specific oligonucleotide probe - A wide variety of formats are available e.g.
dot-blot, Southern blot, reverse hybridization, DNA enzyme immunoassay etc.

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Variations of the basic PCR technique

• Reverse transcriptase PCR (RT-PCR)

• In Reverse Transcriptase or RT-PCR, a strand of RNA is initially reverse transcribed into

its complementary DNA or cDNA using the reverse transcriptase enzyme. The 44

resulting cDNA is further amplified by PCR.

• The cDNA serves as the template for the PCR reaction.
• The RT-PCR is used for detection of RNA viruses in clinical sample and in gene
expression studies.

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Multiplex PCR

• Multiplex PCR refers to the simultaneous amplification of multiple selected target

regions in a sample using different pairs of primers.
• Amplification products are finally differentiated by gel electrophoresis, sequence
specific oligo probes or in a real-time format, by melting curve analysis. 45

• Syndromic approach: for the diagnosis of the infectious diseases- for the
etiological diagnosis of pyogenic meningitis, using different primers targeting
Pneumocooccus, meningococcus and H influenzae ( added simultaneously in the
same reaction tubes)

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Nested PCR
• Nested PCR involves two successive PCRs, where the
amplification product from the first PCR reaction is used
as the template for the second PCR.
• Either one of the primers (semi-nested PCR) or both the
primers (nested PCR) used in the second PCR may be
different from the primers used in the first PCR. 47
• More sensitive (high quantity of DNA), more specific (uses
2 primers targeting 2 regions of DNA of the same
organisms)
• Useful for detection of Mycobacterium tuberculosis
(targeting IS6110) in samples

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Real time PCR

• The Real Time PCR method is used for the

detection and quantitation of an amplified
PCR product as the reaction progresses in
‘real time.’
• incorporation of a fluorescent dye where
the increase in fluorescence signal,
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generated during the PCR, is in direct
proportion to the amount of the PCR
product.
• Here, the fluorescent molecules added to
the PCR mixture produce fluorescent
signals which are detected simultaneously
with the progress in amplification.

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Detection of amplification products of real-time PCR

• Non specific method: SYBR Green that stains any nucleic acid nonspecifically
• Specific methods: they use fluorescent labeled oligonucleotide probe which binds only to
a particular region of amplified nucleic acid. Three types of hybridization probes are
commonly used. 50

• Taqman or hydrolysis probe

• Molecular beacon
• Fluorescence resonance energy transfer (FRET) probe

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Real-time quantitative PCR signal generated from a TaqMan

probe. The normalized fluorescence (ΔRn) is plotted against
PCR cycles 1 to 50. The threshold cycle is indicated by the
green line. The length of the lag phase (number of cycles
required to reach a threshold level of fluorescence) is inversely
correlated with the amount of starting material. 51

Ct values (y axis) were determined for serial 10-fold dilutions of

a synthetic target of known concentration (x axis). The resulting
standard curve is used to convert Ct values of test samples to
concentration.
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Biofire FilmArray
• Automated multiplex nested PCR system, where all the steps from sample preparation to
amplification, detection and analysis are performed automatically by the system
• Excellent sensitivity and specificity
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• Result in one hour.

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PCR for
tuberculosis
• Nested PCR targeting IS6110 gene.
• Other genes targeted- MPT64 gene,
65KDa and 38KDa genes
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• Automated Real Time PCR

• Cartridge-based nucleic acid amplification

(CBNAAT): GeneXpert
• Chip based real time PCR

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GeneXpert
• Cepheid’s CBNAAT system
• Lowest turn around time
• Principle: It is based on real time PCR technique, siultaneously detects
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• MTB complex DNA
• Rifampicin resistance (mutations in the rpoB gene)
• Use 5 probes targeting vrious sequences of rpoB gene
• WHO recommends EPTB diagnostic –initial test, specially for CSF, lymph nodes and other tissue
specimens
• Detection limit: 131 bacilli/mL of specimen
• Expensive and cannot further speciates MTB complex

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