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Joint detection method for drug resistance of mycobacterium tuberculosis and pyrazinamide in clinical sample

A technology of mycobacterium tuberculosis and pyrazinamide, which is applied in the biological field to achieve the effects of improving sensitivity, short detection time, and shortening time

Active Publication Date: 2013-02-13
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the problem of amplification efficiency, this method cannot be well used for the detection of pyrazinamide resistance of Mycobacterium tuberculosis in clinical samples, and can only be used for clinical isolates
But it takes up to a month to isolate M. tuberculosis from a clinical sample

Method used

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  • Joint detection method for drug resistance of mycobacterium tuberculosis and pyrazinamide in clinical sample
  • Joint detection method for drug resistance of mycobacterium tuberculosis and pyrazinamide in clinical sample
  • Joint detection method for drug resistance of mycobacterium tuberculosis and pyrazinamide in clinical sample

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Mycobacterium tuberculosis standard strain M. tuberculosis H37Ra (American Culture Collection Number: ATCC 25177) was used as a pyrazinamide-resistant control, and the standard pyrazinamide-resistant strain BCG (American Culture Collection Number: ATCC 19274) was used as a pyrazinamide-resistant control. The measurement results of these two cultured tuberculosis strains are used to establish a fluorescent quantitative PCR standard curve for judging whether the actual sample contains Mycobacterium tuberculosis and to test whether the method can distinguish standard pyrazinamide drug-resistant strains from sensitive strains.

[0047] Specific steps are as follows:

[0048] 1. Lysis of Mycobacterium tuberculosis and DNA extraction:

[0049] (1) From the cultured standard strain of Mycobacterium tuberculosis M. tuberculosis On the L-J medium of H37Ra and BCG, scrape a ring of bacteria with an inoculation loop, dissolve in 2 mL NaOH solution, mix well, and incubate at ...

Embodiment 2

[0078] In this embodiment, 3 clinical sputum (No. 218, 271, and No. 8333) collected from Wuhan Tuberculosis Prevention and Control Center as test samples are used as test samples to test whether this method can be used for the classification of tuberculosis in clinical sputum. The detection of mycobacteria and the detection of pyrazinamide resistance were initially verified. Specific steps are as follows:

[0079] 1. Cracking and extracting the DNA of Mycobacterium tuberculosis in sputum;

[0080] (1) Add 2 mL of NaOH solution to a certain amount of sputum (1-5 mL), mix well, and incubate at 37°C for 20 minutes.

[0081] (2) Centrifuge for 10 min, remove the supernatant and retain the precipitate. Wash with PBS, repeat the wash 2 times, remove the supernatant and save the precipitate for detection.

[0082] (3) DNA extraction: Add DNA extraction solution and purified water to the above-mentioned processed specimens. Boiling water bath for 10 min, and then directly use the ...

Embodiment 3

[0103] In the step of pyrazinamide resistance detection, it is necessary to add in vitro expression elements, such as T7 promoter, expression enhancer, etc., to the fluorescent quantitative PCR product. The present embodiment uses pyrazinamide sensitive strain Mycobacterium tuberculosis standard strain M. tuberculosis H37Ra (American Culture Collection Number: ATCC 25177) was used as a pyrazinamide-resistant control, and the standard pyrazinamide-resistant strain BCG (American Culture Collection Number: ATCC 19274) was used as a pyrazinamide-resistant control. The strains were used for in vitro expression PCR with and without primers of an expression enhancer 5' UTR. Then it was added to the wheat germ cell-free expression system to study whether the expression enhancer in the primer would affect the determination of pyrazinamide resistance.

[0104] Specific steps are as follows:

[0105] 1. adopt the same method as in Example 1 to lyse Mycobacterium tuberculosis standard...

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Abstract

The invention relates to a joint detection method for the drug resistance of mycobacterium tuberculosis and the pyrazinamide thereof in a clinical sample, comprising the following steps of: A, extracting DNA (deoxyribonucleic acid) in the sample; B, detecting the mycobacterium tuberculosis via fluorescent quantitative PCR (polymerase chain reaction) amplification for a tuberculosis genome segment containing total-length pyrazinamide enzyme genes (pncA); and C, detecting the drug resistance of pyrazinamide for the mycobacterium tuberculosis positive sample, wherein the steps for detecting the drug resistance of the pyrazinamide are as follows: 1, performing PCR reaction once by taking the fluorescent quantitative PCR product as a template, so as to add an in-vitro expression element; 2, adding the PCR product in a cell-free expression system, so as to express a pyrazinamide enzyme; and 3, comparing the pyrazinamide enzyme activity of the sample with a standard strain, so as to give the drug resistance result of pyrazinamide. According to the invention, joint detection for the drug resistance of mycobacterium tuberculosis and the pyrazinamide thereof in the sample is realized by combining fluorescent quantitative PCR amplification for pncA genes with the enzyme activity of the in-vitro expressed pyrazinamide enzyme, without the need of bacterial culture and a DNA sequencer.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for combined detection of drug resistance of Mycobacterium tuberculosis and pyrazinamide in clinical samples based on fluorescent quantitative PCR and cell-free in vitro protein expression system, which can be used for detecting and confirming clinical samples Mycobacterium tuberculosis and its pyrazinamide resistance in . Background technique [0002] Tuberculosis is a major infectious disease that threatens human health. The drugs currently used for first-line treatment include pyrazinamide, isoniazid, rifampicin and ethanol. With the promotion of drug chemotherapy, a large number of drug-resistant tuberculosis bacteria also appeared, which greatly affected the treatment of tuberculosis patients. Effective diagnosis of tuberculosis and detection of drug resistance are of great significance for guiding clinical treatment of tuberculosis. But at present, both as...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/34C12Q1/04C12R1/32
Inventor 危宏平李恒周满耿学磊王殿冰余军平张先恩
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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