Sansure Biotech

Mycobacterium Tuberculosis DNA Fluorescence Diagnostic Kit 8 TB-Negative Control 50 μL/tube × 1 tube Normal saline

(PCR-Fluorescence Probing) 9 TB-Positive Control 50 μL/tube × 1 tube Quantitative TB positive specimen (inactivated)

Positive Internal Reference (Cloning Plasmid, without TB

10 TB-Internal Control 50 μL/tube × 1 tube
【 Reference Number 】 target sequence)
S3018E Note:
【 Product Name 】 1. Do not mix or exchange components from different kit lots.
Mycobacterium Tuberculosis DNA Fluorescence Diagnostic Kit (PCR-Fluorescence Probing) 2. All biological specimens in the detection kit should be handled as infectious though they have been inactivated.
【 Package Specification 】 3. Self-prepared reagent: Normal saline and 4% NaOH solution.
48 tests/kit 4. Processing of specimen: Customers may use Sansure Biotech ’s magnetic-bead method based extraction reagent to process
【 Intended Use 】 specimen, then the DNA doesn’t need to be treated by lysis buffer.
This diagnostic kit is an in vitro nucleic acid amplification test for the detection of mycobacterium tuberculosis (TB) DNA in human 5. Materials required but not provided: 1.5 mL DNase-free and RNase-free centrifuge tubes, 0.2 mL PCR reaction tubes, pipette
sputum. It is intended for use as an aid in the diagnosis of a TB infection. tips (10 μL, 200 μL and 1000 μL tips with filters are preferred), desktop centrifuge, desktop vortex mixer, magnetic-bead separator,
various models of pipette guns.
For in vitro diagnostic use only. For professional use only. 【 Storage 】
【 Summary 】 The detection kit should be stored in a sealed pouch at -20±5°C and protected from light. The shelf life of the kit is 12 months.

Mycobacterium tuberculosis virus (TB) is a pathogenic bacterium that causes tuberculosis. It is likely to infect all human tissues and Multiple freeze/thaw cycles should be avoided.

organs, especially the lungs to cause pulmonary tuberculosis. Early diagnosis and treatment are important for effective control of 【 Compatible Instrument 】

tuberculosis. In recent years, with the development of molecular biology, nucleic acid fluorescence quantitative PCR method based The diagnostic kit is applicable to fluorescence PCR instruments such as ABI7500, Stratagene Mx3000P.

on the mycobacterium tuberculosis nucleic acid has drawn more and more attention from researchers. 【 Specimen Requirements 】

【 Test Principle 】 1. Applicable specimen type: human sputum.

The diagnostic kit uses a nucleic acid lysis buffer to allow rapid lysis and release of TB-DNA from a sputum specimen. By applying 2. Collection of specimen:

real-time fluorescence quantitative PCR technology, this test uses a pair of specific primers which are designed to target a It is recommended to collect the first sputum in the morning. First rinse mouth with water. Make a hard cough and collect the sputum

conserved sequence of TB-DNA and a specific fluorescence probe, accompanied with other ingredients in PCR mix, to achieve fast in the deep and keep it in a sterile collection tube. Seal it and send it for detection.

detection of TB-DNA through fluorescent signal changes. 3. Storage and delivery of specimens:

The PCR detection system uses UNG enzyme + dUTP contamination-proof system, which can fully degrade possible unwanted Specimens collected via the above-mentioned method can be used for immediate detection, or stored at 2-8°C for up to 24 hours, or

side-products, to avoid a false positive result. below -20°C for a longer term of storage. Caution should be taken to avoid re-freezing and re-thawing. Specimens should be

【 Components of the Diagnostic Kit 】 transported in a sealed frozen pitcher with ice or in a sealed foam box with ice.
【 Test Method 】
No. Reagent Name Specification & Qty. Main Ingredients
1. Preparation of reagent (performed at “reagent preparation region”)
1 TB-Lysis Buffer 2.5 mL/tube × 1 tube KCl, SDS, surfactin 1.1 Take out each component from the detection kit and place them at room temperature. When the components’ temperature has

2 DNA polymerase, uracil, DNA glycosylase reached room temperature, mix them for later.
TB-Enzyme Mix 96 μL/tube × 1 tube
1.2 Refer to quantities of test specimens, TB-Negative Control, TB-Positive Control, and TB-Positive References A-D, pipette
3 TB-PCR Mix 912 μL/tube × 2 tubes primer, probe, dNTPs, Mg2+, PCR buffer solution
appropriate quantities of TB-PCR Mix, TB-Enzyme Mix and TB-Internal Control (TB-PCR Mixx 38 μL/test + TB-Enzyme Mix 2
TB-Positive Reference A μL/test + TB-Internal Control 1 μL/test), fully mix them to make a PCR-Mastermix and then centrifuge it instantaneously for later.
4 50 μL/tube × 1 tube Cloning plasmid with target gene fragment
(4.00E+07 copies/mL) 1 sample 10 samples 24 samples 48 samples
TB-PCR Mix (μL) 38 380 912 1824
TB-Positive Reference B
5 50 μL/tube × 1 tube Cloning plasmid with target gene fragment TB-Enzyme Mix (μL) 2 20 48 96
(4.00E+06 copies/mL)
TB-Internal Control (μL) 1 10 24 48
TB-Positive Reference C Note: The above configuration is just for your reference and to ensure enough volume of
6 50 μL/tube × 1 tube Cloning plasmid with target gene fragment
(4.00E+05 copies/mL) the PCR-Mastermix, more volume of the actual pipetting may be required.
2. Processing and Loading of specimens (performed at “specimen processing region”)
TB-Positive Reference D
7 50 μL/tube × 1 tube Cloning plasmid with target gene fragment 2.1 Processing of specimen
(4.00E+04 copies/mL)
Add 4% NaOH solution into specimen with the solution volume of 2-3 times to specimen. Vortex it and hold it for 30 minutes to allow

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it to liquefy. Aspirate 500 μL of liquefied specimens to a 1.5 mL centrifuge tube (avoid aspirating apparent solid impurities). is determined to be 40.
Centrifuge it at 12000 rpm for 3 minutes. Aspirate and discard the supernatant. Add 1 mL of sterile saline and resuspend the 【 Explanation of Detection Result 】
precipitate. Centrifuge it at 12000 rpm for 3 minutes. Aspirate and discard the supernatant. Add 50 μL of nucleic acid lysis buffer. Conclusion Ct value of sample Ct value of internal control Amplification curve of sample
Process it at 100°C for 10 minutes. Centrifuge it at 12000 rpm for 3 minutes. Take the supernatant as the under-test specimen for Positive ≤ 39 - like "S"
later. Negative N/A ≤ 40 like "--"
2.2 Loading of specimens (negative control and specimens are processed together) Out of limit
2.2.1 Add 5 μL of under-test specimen, TB-Negative Control, TB-Positive Control and TB-Positive Reference A-D respectively into (concentration < 4.00E+02 >39 ≤ 40 like "S"
each PCR reaction tube. copies/mL)
2.2.2 Add 40 μL of PCR-Mastermix into each tube. Cover the tube (after removing the bubbles). Centrifuge it at 2000 rpm for 30 Invalid* - > 40 or N/A -
seconds. Note: *This suggests an investigation should be carried out to find out the reasons when Ct value of the internal control is >
3. PCR Amplification (performed at “amplification and analysis region”; refer to user manual of each instrument to adjust the 40 or N/A and retest it. (If repeated tests still produce invalid results, please contact Sansure Biotech at
settings) [email protected])
3.1 Place the PCR reaction tube into the specimen wells of the amplification device. Set up the negative control, positive control,
This kit is only used for qualitative detection of mycobacterium tuberculosis (TB) DNA. The quantitative results can only be used for
positive references A-D and unknown samples in the corresponding sequence and input sample information.
reference and cannot serve as a basis for clinical report.
3.2 Select PCR test channel
【 Limitations of Detection Method 】
For ABI, Stratagene series:
Detection result is related to specimen collection, processing, delivery and storage quality. Any deviation from the stated procedure
a. Select FAM channel (Reporter: FAM, Quencher: None) to test TB-DNA.
will lead to an inaccurate detection result. Cross-contamination during specimen processing may also result in a false-positive
b. Select HEX or VIC channel (Reporter: HEX/VIC, Quencher: None) to test TB-Internal Control.
result.
c. Set passive reference: none.
【 Product Performance Index 】
d. Set sample volume: 50.
When the kit is used to detect the enterprise work references, the consistency rate for both negative and positive reaches 100%.
3.3 Set cycle parameters (the time parameter varies according to instruments):
Precision test shows excellent reproducibility in both intra-batch and inter-batches with its coefficient of variation of Ct value < 10%,
For ABI, Stratagene series:
and its coefficient of variation of concentration < 50%. The sensitivity of this kit is 1 bacterium/mL. It shows no cross-reaction with
Step Temperature Time Cycle No. Mycobacterium Avium, Land Mycobacterium, Amur Mycobacterium, Mycobacterium Kansasii, Asian Mycobacterium,
M.scrofulaceum Mycobacterium, Gordon Mycobacterium, Turtle pus Mycobacterium, Accidental Mycobacterium, Mycobacterium
1 UNG enzyme reaction 50°C 2 min. 1
phlei, Brazilian Nocardia, Corynebacterium pekinense, pneumococcus, legionella pneumophilia, Bordetella pertussis, MP, EBV and
2 Taq enzyme activation 94°C 5 min. 1 respiratory syncytial virus, etc.
Denaturation 94°C 15 sec. 【 Precautions 】
3 45
Annealing, extension, fluorescence collection 57°C 30 sec.* 1. The product can only be used for in vitro diagnosis. Please read the product manual carefully before operation.
4 Device cooling (optional) 25°C 10 sec. 1 2. Please learn and be familiar with the operation procedures and precautions for each instrument before test. Please make sure
Note: Due to the device ABI 7500’s technical specification, it can not be set at 30 seconds but 31 seconds. quality control for each test.
When the settings are completed, save settings and carry out the reaction procedure. 3. Laboratory management shall strictly follow management practices of PCR gene amplification laboratory; laboratory personnel
4. Result Analysis (Refer to user manual of each instrument to adjust the settings) must receive professional training; test processes must be performed in separated regions; all consumables should be for single
When the reactions are completed, results will be saved automatically. After analysis, adjust Start, End and Threshold values of use only after sterilization; special instruments and devices should be used for every process; all lab devices used in different
Baseline of the graph (users can adjust them according to the actual situations. Start value can be set at 3-15, and End value at processes and regions should not be cross-used.
5-20. Adjust the amplification curve of negative control to be flat or below threshold). Click “Analyze” to implement the analysis and 4. All specimens for detection should be handled as if infectious. Wear laboratory coats, protective disposable gloves and change
make sure each parameter satisfy the requirements given in “5. Quality Control”. Go to “Plate” window to record the detected Ct the gloves often to avoid cross-contamination between samples. Handling of specimens and waste must meet relevant
value. requirements outlined in “Biosafety Common Criteria in Microbiological and Biomedical Laboratories” and “Medical Waste
5. Quality Control Management Regulations” by Ministry of Health.
The test result is treated as valid if all the conditions in the table below are met for the same test. Otherwise the test result is treated 5. Before use, all reagents must be fully thawed at room temperature and mixed thoroughly.
as invalid and needs to be re-tested. 【 Bibliography 】
Positive Control Negative Control Internal Control Positive References (A, B, C, D) 1. Benjamin A. Pinsky ,Niaz Banaei.Multiplex Real-Time PCR Assay for Rapid Identification of Mycobacterium tuberculosis Complex
Ct value ≤ 30 N/A ≤ 40 ≤ 39 Members to the Species Level.Journal of Clinical Microbiology, 2008, 46:2241-2246.
2. Marion Blaschitz1,Dzenita Hasanacevic1,Peter Hufnagl,et al.Real-time PCR for single-nucleotide polymorphism detection in the 16S
【 Reference Range 】
rRNA gene as an indicator for extensive drug resistance in Mycobacterium tuberculosis.J. Antimicrob. Chemother,2010,45:1093-1110
Through the research on reference values, the Ct reference value of target gene is 39, and the Ct reference value of internal control

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3. van Doorn HR, An DD, de Jong MD, Lan NT, et al.Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked
nucleic acid probe real-time PCR.Int J Tuberc Lung Dis. 2008(7):736-42.
4. B.J. Renton, P. D. Morrell.Direct real-time PCR examination for Mycobacterium tuberculosis in respiratory samples can be cost
effective.Health,200 9,11:63-66.
【 Symbols 】

Symbols Meanings Symbols Meanings

Date of Manufacture
In Vitro Diagnostic Medical Device
For Professional Use Only

Use By Consult Instructions for Use

Temperature Limitation Manufacturer

Lot Number Reference Number

Authorized representative in
Number of Tests
the European Community

This product fulfills the

Any warnings and/or precautions to requirements of the European
take Directive 98/79 EC for in vitro
diagnostic medical devices.

Sansure Biotech Inc.

Add.: No. 680, Lusong Road, Yuelu District, 410205 Changsha, Hunan Province,

PEOPLE’S REPUBLIC OF CHINA

Tel.: +86-731-88883176

Fax: +86-731-88884876

Web: www.sansure.com.cn

Obelis S.A

Bd. Général Wahis 53, 1030 Brussels, BELGIUM

Tel: + (32) 2.732.59.54

Fax: + (32) 2.732.60.03

E-Mail : [email protected]

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