Pyrosequencing-based method for detecting tolerance of mycobacterium tuberculosis ethambutol

A technology of Mycobacterium tuberculosis and pyrosequencing, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem of no relevant reports on the detection of hexambutol, and achieves simple operation, good sensitivity and specificity. degree of effect

Inactive Publication Date: 2014-06-18
山东国际旅行卫生保健中心
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Problems solved by technology

[0004] Pyrosequencing technology is a new type of DNA sequencing technology. Compared with Sanger sequencing method, which is the gold standard of molecular drug susceptibility, it does not need to measure the entire gene, but only needs to sequence specific short fragments to detect all type...

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  • Pyrosequencing-based method for detecting tolerance of mycobacterium tuberculosis ethambutol
  • Pyrosequencing-based method for detecting tolerance of mycobacterium tuberculosis ethambutol
  • Pyrosequencing-based method for detecting tolerance of mycobacterium tuberculosis ethambutol

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Embodiment Construction

[0049] The content of the present invention will be further described in detail below through examples, so as to better understand the content of the present invention, but the following examples do not limit the protection scope of the present invention.

[0050] DNA extraction

[0051] The strain was inoculated on the modified Roche medium, cultured at 37°C for 2-3 weeks, and the MTB genomic DNA was extracted using the bacterial lysate produced by Takara Company. The specific operation is as follows: wash the fresh culture of the bacterial strain with sterile physiological saline, draw 1ml of the bacterial suspension into a sterile Eppendorf centrifuge tube, take 50 μl of lysate into a centrifuge tube, and shake it on a vortex shaker until the blocky solid colonies are evenly dissolved. After thermal denaturation at 80°C for 15 minutes in the solution, centrifuge at 12,000 r / min for 3 minutes, take 1-5 μl of the lysed supernatant as a PCR reaction template or freeze it at -2...

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Abstract

The invention discloses a pyrosequencing-based method for detecting tolerance of mycobacterium tuberculosis ethambutol. The method comprises the following steps: 1, carrying out polymerase chain reaction (PCR) amplification on an embB gene; designing a PCR amplification primer aiming at 306 sites of the embB gene; taking a sample mycobacterium tuberculosis deoxyribonucleic acid (DNA) extract as a template, and carrying out PCR amplification on the embB gene containing the 306 sites of the embB gene, wherein the length of a DNA segment for amplifying the mycobacterium tuberculosis embB gene is 165bp, and the sequence containing the 306 sites of the embB gene is atg; 2, carrying out pyrosequencing on the PCR amplification product, judging whether the 306 sites of the embB gene have mutation or not, wherein the pyrosequencing adopts a supplier quality assurance (SQA) mode, and the nucleotide loading order is AGCT. By check for a plurality of times, the drug-resistant mutation site of ethambutol embB gene can be accurately detected by a test primer of the shown sequence, the drug-resistant strains of the mycobacterium tuberculosis can be quickly screened, and early detection, early diagnosis and early treatment of a sufferer infected with the drug-resistant mycobacterium tuberculosis are achieved.

Description

technical field [0001] The invention relates to a method for detecting drug resistance of Mycobacterium tuberculosis, in particular to a method for detecting drug resistance of Mycobacterium tuberculosis based on pyrosequencing. Background technique [0002] Tuberculosis is a chronic infectious disease that seriously endangers human health, and is currently the number one killer of infectious diseases threatening human health. The generation and dissemination of drug-resistant tuberculosis strains, especially the emergence of multidrug-resistant tuberculosis strains, has become one of the three major problems in tuberculosis control in the new century. Rapid and accurate detection of MDR-TB is of great significance for early detection, blocking the spread of drug-resistant TB, standardizing and guiding drug use, and improving the cure rate of drug-resistant TB. [0003] Mycobacterium tuberculosis grows slowly. The traditional detection methods for drug resistance of Mycobac...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/04C12Q1/6869C12Q1/689C12Q2600/106C12Q2600/156C12Q2531/113C12Q2565/301
Inventor 陈晓光程琳郭宁张瑾姜华金燕梁洁张娟
Owner 山东国际旅行卫生保健中心
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