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Method for detecting multi-drug resistance of Mycobacterium tuberculosis

A technology for Mycobacterium tuberculosis and multidrug resistance, applied in the biological field, can solve the problems of PCR-SSCP time-consuming and labor-intensive, the number of MTB resistance genes is large, and the analysis is troublesome.

Inactive Publication Date: 2014-04-16
孙爱华 +2
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Problems solved by technology

In 2007, Hong Kong Chan et al. reported the correlation between certain gene mutations and drug resistance of MTB by using PCR-SSCP. In 2007, Bao Hong et al. used PCR-SSCP to detect the correlation between rpoB mutations and drug resistance in sputum samples. The correlation, specificity and sensitivity of drugs have good clinical significance; however, due to the large number of MTB drug resistance genes, it is still very troublesome and time-consuming to perform PCR-SSCP analysis one by one; domestic Cheng Xiaodong et al. reported that mPCC-SSCP simultaneously Detect three gene mutations related to isoniazid resistance, which solves the time-consuming and labor-intensive defect of a single PCR-SSCP, but only detects a single drug isoniazid resistance-related gene mutation; it does not adapt to the multi-drug resistance of MTB that is increasing year by year Rate

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  • Method for detecting multi-drug resistance of Mycobacterium tuberculosis
  • Method for detecting multi-drug resistance of Mycobacterium tuberculosis
  • Method for detecting multi-drug resistance of Mycobacterium tuberculosis

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Embodiment

[0057] The source of the specimen in this embodiment: the cultured and identified clinical strain of Mycobacterium tuberculosis, or the sputum specimen of a suspected tuberculosis patient.

[0058] Sources of reagents in this example: All experimental materials, reagents and related equipment used in this example can be purchased from respective domestic and foreign related industry companies or domestic sales agents of foreign companies. Professionals in technical fields such as microbiology and molecular biology can repeat the preparation method in this example and try it out.

[0059] 1. DNA genome extraction: Extract from the DNA genomes of 134 cases of MTB clinical strains and standard strain H37Rv that have been cultured and identified; specifically: transfer the bacterial culture into a 1.5ml centrifuge tube, wash with normal saline 3 times, 6000r / centrifuge at m (4°C) for 5 min, discard the supernatant, and extract DNA from the precipitate using a DNA extraction kit, ...

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Abstract

The invention relates to a method for detecting multi-drug resistance of Mycobacterium tuberculosis, which aims at detecting the resistance of the Mycobacterium tuberculosis to isoniazid and rifampicin at the same time and has the characteristics of high specificity and sensitivity, quickness in detection, and easiness and convenience in operation. The technical scheme is as follows: the method comprises the following steps: A, establishing PCR (polymerase chain reaction) templates of an MTB clinical strain and a standard strain H37Rv; B, designing three pairs of primers of katG, inhA and rpoB gene fragments which are closely related to the isoniazid resistance and the rifampin resistance, performing mPCR amplification on katG, inhA and rpoB genes of the clinical strain, and performing PCR amplification on katG, inhA and rpoB genes of the clinical strain and the standard strain H37Rv; and C, detecting the mutation conditions of related INH-resistant and RFP-resistant 3 genes of the clinical strain at the same time by a single-strand conformation polymorphism detection method.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method based on multiple polymerase reaction-single-strand conformation polymorphism (mPCR-SSCP) for simultaneous detection of isomycin resistance of clinical isolates of Mycobacterium tuberculosis (MTB for short). The method for the gene mutation related to hydrazine and rifampicin can be used for rapid detection of isoniazid and rifampicin resistance of clinical Mycobacterium tuberculosis at the same time. Background technique [0002] The number of tuberculosis patients in China ranks second in the world. In recent years, the drug resistance of Mycobacterium tuberculosis has shown a significant growth trend. China is also one of the countries with the highest degree of drug resistance. The emergence of drug-resistant tuberculosis strains has become a serious public health problem. Isoniazid (INH), rifampicin (RFP), streptomycin (SM) and ethambutol (EMB) are clinical first-line a...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/32
Inventor 孙爱华严杰李召东
Owner 孙爱华
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