Method for detecting resistant mutation of mycobacterium tuberculosis to rifampin and kit thereof

A technology for Mycobacterium tuberculosis and drug-resistant mutations, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, and material stimulation analysis, etc. It can solve the problems of increased PCR product contamination and cumbersome detection steps.

Inactive Publication Date: 2011-11-02
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above methods are different in detection rate and throughput, but the common disadvantage is that there is a PCR po

Method used

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  • Method for detecting resistant mutation of mycobacterium tuberculosis to rifampin and kit thereof
  • Method for detecting resistant mutation of mycobacterium tuberculosis to rifampin and kit thereof
  • Method for detecting resistant mutation of mycobacterium tuberculosis to rifampin and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Extraction of Mycobacterium tuberculosis DNA

[0084] 1) Culture of Mycobacterium tuberculosis strain

[0085] Mycobacterium tuberculosis was cultured in acidic Roche medium.

[0086] 2) DNA extraction

[0087] DNA was extracted from samples of Mycobacterium tuberculosis strains by boiling and lysis. Specific steps are as follows:

[0088] A. For Mycobacterium tuberculosis growing on solid medium, use 22SWG standard inoculation loop to collect 1 loop of bacteria, and suspend in 250 μL TB DNA extraction solution. Take 1 mL of Mycobacterium tuberculosis grown in liquid medium, centrifuge at 10,000 rpm for 15 min, discard the supernatant and resuspend the bacteria in 250 μL of TB DNA extract.

[0089] B. Seal with parafilm and heat at 99°C for 20 minutes. Centrifuge at 14000rpm for 10min, transfer the supernatant to a new 1.5mL centrifuge tube. The supernatant is the template for PCR amplification.

[0090] C. Samples should be stored at -20°C and the test...

Embodiment 2

[0091] The design of embodiment 2 Mycobacterium tuberculosis detection primers and probes

[0092] According to the rpoB (GenBank: L27989) sequence of the rifampicin resistance-related gene of Mycobacterium tuberculosis, it was completed with software such as Primer 5, TmUtility v1.3 and mfold. The primer and probe sequences are as follows:

[0093] Primer-F: 5′-GGAGGCGATCACACCGCAGACGTT-3′

[0094] Primer-R: 5′-TGACAGACCGCCGGGCC-3′

[0095] Probe A: 5'-FAM-CGAGCTCAGCTGGCTGGTGCGCTCG-BHQ1-3'

[0096] Probe B: 5'-FAM-GCTACGGAGCCAATTCATGGACCAGACGTAGC-BHQ1-3'

[0097] Probe C: 5'-TET-CCGACGCCGACAGCGGGTTGTTCGTCGG-BHQ1-3'

[0098] Probe D: 5'-TET-CCTGCCGCCGACTGTCGGCGCTGGCAGG-BHQ1-3'.

[0099] Primers and probes were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Store the probes in the dark after quantification.

Embodiment 3

[0100] Example 3 Mycobacterium tuberculosis rifampicin-resistant mutation detection kit detects rifampicin-resistant mutation

[0101] Two reaction systems are used for double-color detection:

[0102] Each part of RIF PCR MIX A is 19.6μl, each reaction solution contains 1×PCR buffer (10mM Tris-HCl, pH8.6, 50mM KCl, 50% glycerol), 3.0mM MgCl 2 , dATP, dCTP, dGTP each 0.2mM, dUTP 0.4mM, 0.1μM Primer-F, 2μM Primer-R, 0.1μM Probe-A, 0.1μM Probe-C. 2U of enzyme mixture (0.4 μl) was added before detection, and the amount of template to be tested was 5 μl.

[0103] Each part of RIF PCR MIX B is 19.6μl, and each reaction solution contains 1×PCR buffer (10mM Tris-HCl, pH8.6, 50mM KCl, 50% glycerol), 3.0mM MgCl 2 , dATP, dCTP, dGTP each 0.2mM, dUTP 0.4mM, 0.1μM Primer-F, 2μM Primer-R, 0.1μM Probe-B, 0.1μM Probe-D. 2U of enzyme mixture (0.4 μl) was added before detection, and the amount of template to be tested was 5 μl.

[0104] Take the concentration as 10 4 Copies / μl RIF wild-ty...

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Abstract

Relating to the detection technology of resistant mutation of mycobacterium tuberculosis, the invention aims to provide a rapid, sensitive and specific method for detecting the resistant mutation of mycobacterium tuberculosis to rifampin and a kit thereof. The method comprises the steps of: extracting the DNA of a mycobacterium tuberculosis sample; according to the rpoB gene sequence (GenBank: L27989) where an RRDR (rifampin resistance determining region) of mycobacterium tuberculosis is located, designing primers and probes by means of the primer design software Primer Premier 5; constructing a double-tubed and double-colored real-time PCR (polymerase chain reaction) system, detecting the mutation of the RRDR of mycobacterium tuberculosis, comparing the melting point (Tm value) differences of melting curves between the detected sample and a positive control, and determining whether the sample mutates. With the primers designed at two ends of the RRDR of the rpoB gene, the method of the invention is of high specificity.

Description

technical field [0001] The invention relates to a detection technology for drug-resistant mutations of mycobacterium tuberculosis, in particular to a probe melting curve analysis technology based on double-labeled self-quenching probes for detecting rifampin drug-resistant mutations of mycobacterium tuberculosis. Background technique [0002] Since the 1980s, the epidemic of tuberculosis has risen again, and it has become the most serious infectious disease in the world alongside AIDS. According to the statistics of the World Health Organization, one-third of the world's population is currently infected with Mycobacterium tuberculosis, with 8 million new patients and 2 to 3 million deaths each year. It is currently the infectious disease with the highest mortality rate. According to the assessment of the World Health Organization, my country is one of the 22 countries with a high burden of tuberculosis in the world, and the number of patients is second only to India, ranking...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02G01N21/64
Inventor 李庆阁胡思玉张轶刘歆
Owner XIAMEN UNIV
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