Method for detecting mycobacterium tuberculosis rpoB mutant genes

A technology for Mycobacterium tuberculosis and mutated genes, which is applied in the detection of Mycobacterium tuberculosis rpoB mutant gene and the detection of rifampicin resistance of Mycobacterium tuberculosis, can solve the problems that cannot meet the requirements of clinical diagnosis, the sequencing method is expensive, and the The problem of high pollution probability can improve the detection rate and identification accuracy, shorten the detection cycle, and achieve the effect of good map identification.

Inactive Publication Date: 2012-11-21
NANYANG MEDICAL COLLEGE
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Problems solved by technology

[0006] The existing methods used for the detection of rifampicin resistance of Mycobacterium tuberculosis include absolute concentration method, RFLP method and sequencing method. Among them, the absolute concentration method has secondary culture, eight weeks each time, long cycle and contamination probability High; du

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  • Method for detecting mycobacterium tuberculosis rpoB mutant genes
  • Method for detecting mycobacterium tuberculosis rpoB mutant genes

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Embodiment

[0027] Examples, see figure 1 A method for detecting the rpoB mutant gene of Mycobacterium tuberculosis of the present invention adopts the following steps: culturing-extracting-amplifying-purifying-digesting-separating-dense-imaging-identification, wherein:

[0028] A. Culture, collect the sputum of tuberculosis patients, inoculate it into the modified Roche medium (provided by a certain pharmaceutical company in Wuxi), and culture it in a 37°C incubator for 4 weeks at a time;

[0029] B. Extraction, take a circle of Mycobacterium tuberculosis culture and dissolve it in 100μl distilled water, heat at 100°C for 30 minutes, centrifuge at 12,000g for 10 minutes, take 2.5μl of the supernatant as the DNA template in the PCR reaction system, and store the rest in - Store in the refrigerator at 20°C for later use;

[0030] C. Amplification, using the following reaction system to carry out PCR amplification reaction:

[0031] 10×Taq Buffer 5.0μl, 10mmol / L dNTP 4.0μl, 20mmol / L novel...

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Abstract

The invention belongs to the field of molecular biology detection, relates to a mycobacterium tuberculosis rifampicin drug resistance detection method, and provides a method for detecting mycobacterium tuberculosis rpoB mutant genes. The method comprises the following steps of culture, extraction, amplification, purification, digestion, separation, gathering, image displaying and identification. Compared with the existing similar detection methods, the method of the invention adopts once culture, novel forward and backward primers are adopted, the purification step is adopted, the cracking digestion is adopted, the gathering step is adopted before the image displaying, a labeled digoxin antibody developing system is adopted, conventional equipment is adopted, and medicament can be easily obtained, so the method provided by the invention has the obvious advantages that the detection is fast, the detection rate is high, the map identification performance is good, the identification is accurate, the cost is low, and the like. The clinic diagnosis and treatment requirements can be more exactly and efficiently met.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, relates to a detection method for rifampicin drug resistance of mycobacterium tuberculosis, and provides a method for detecting rpoB mutation gene of mycobacterium tuberculosis. Background technique [0002] In recent years, both developed and developing countries have seen a big rebound in the prevalence of tuberculosis worldwide. According to statistics, 1 / 3 (about 2 billion) people in the world have been infected with tuberculosis. There are about 20 million tuberculosis patients, about 9 million new patients occur every year, and the annual death toll is as high as 3 million. The emergence of multidrug-resistant tuberculosis (MDR-TB) / extensively drug-resistant tuberculosis (XDR-TB) poses a serious threat to global public health security and greatly increases the global tuberculosis control. burden. According to WHO data, the number of drug-resistant tuberculosis patients in China ...

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Application Information

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IPC IPC(8): C12Q1/68C12R1/32
Inventor 满永宏包东武罗小萌
Owner NANYANG MEDICAL COLLEGE
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