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Method and kit for detecting multi-drug resistant mycobacterium tuberculosis (MDR-TB)

A Mycobacterium tuberculosis, drug-resistant technology, applied in biochemical equipment and methods, DNA/RNA fragments, material excitation analysis, etc., can solve problems such as increasing method specificity and increasing detection costs

Active Publication Date: 2010-09-29
无锡锐奇基因生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because of the use of two probes in the reaction, the specificity of the method is increased, but the detection cost is also greatly increased

Method used

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  • Method and kit for detecting multi-drug resistant mycobacterium tuberculosis (MDR-TB)
  • Method and kit for detecting multi-drug resistant mycobacterium tuberculosis (MDR-TB)
  • Method and kit for detecting multi-drug resistant mycobacterium tuberculosis (MDR-TB)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Design and synthesis of primers and probes

[0073] Rifampicin and isoniazid are the key first-line drugs in the tuberculosis treatment regimen and the anti-tuberculosis drugs most prone to drug resistance. Studies have shown that 97% of rifampicin-resistant strains are caused by mutations in rpoB genes (511, 513, 515, 516, 518, 519, 522, 526, 531 and 533 sites), and the mutations are mainly concentrated in a highly conserved 81bp region. Isoniazid resistance is mainly related to the katG gene (position 315) encoding catalase-peroxidase, the inhA gene encoding reduced nicotinamide adenine dinucleotide-dependent enoyl carrier protein reductase ( -8, -15) and mutations in the ahpC gene (-6, -9, -10 and -12) encoding alkyl catalase. The embB306 mutation is a highly specific genetic marker associated with tuberculosis multidrug resistance. Design primers and probes based on the above five gene sequences related to tuberculosis drug resistance, and ensure that ...

Embodiment 2

[0076] Embodiment 2: the extraction of sample DNA

[0077] The clinical sample isolates identified as positive for Mycobacterium tuberculosis after culture were taken, inactivated by high temperature, and then the sample DNA was extracted with a commercial genomic DNA extraction kit. The obtained genomic DNA was directly used for tuberculosis drug resistance detection or stored at -20°C until use.

Embodiment 3

[0078] Embodiment 3: Extraction of sample DNA

[0079] Take 2ml of sputum, add 2.5 times the volume of 4% NaOH, and incubate at 37°C for 30min. The liquefied sputum was centrifuged to remove the supernatant, and the resulting precipitate was used for sample DNA extraction. Sample DNA was extracted with a commercial genomic DNA extraction kit. The obtained genomic DNA was directly used for tuberculosis drug resistance detection or stored at -20°C until use.

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Abstract

The invention relates to a method adopting double-label probe detection and melting curve analysis for diagnosing the infection of multi-drug resistant mycobacterium tuberculosis and a kit which utilizes the method to detect multiple gene mutations related to drug resistant tuberculosis at the same time, and the invention belongs to the life science and biological technical field. The kit of the invention contains a primer designed for multiple gene mutations related to drug resistant, a double-label oligonucleotide probe capable of detecting multiple common gene mutation sites related to drug resistant tuberculosis and a DNA polymerases with heat stability and without 5' nuclease activity, and the kit can be used to detect at least 16 common gene mutation sites related to drug resistant tuberculosis under proper PCR reaction conditions. The detection method and kit of the invention can be used in the early diagnosis of multi-drug resistant tuberculosis and overcome the problems of the existing technology that the detection time is long, a great deal of manpower and large material resources are needed, the detection cost is high, etc.

Description

technical field [0001] The present invention relates to a medical in vitro diagnostic technology for microbial detection, in particular to a detection method for multidrug-resistant Mycobacterium tuberculosis infection, and a kit for simultaneously detecting multiple gene mutations related to tuberculosis drug resistance by using the method . Background technique [0002] Tuberculosis remains a serious public health problem, especially in developing countries. According to WHO statistics, 1 / 3 of the world's population is infected with Mycobacterium tuberculosis (MTB), which is the second largest killer of infectious diseases in the world. And in recent years, the drug resistance of Mycobacterium tuberculosis has shown a significant growth trend. About 4.3% of new cases and treated patients in the world have multi-drug resistance, and the multi-drug resistance rate in some high-incidence areas is as high as 10%. my country is one of the countries with severe tuberculosis e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64C12N15/11
Inventor 富国良罗涛姜丽丽高谦
Owner 无锡锐奇基因生物科技有限公司
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