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Primer set for detection of Mycobacterium tuberculosis and its resistance to pyrazinamide

A Mycobacterium tuberculosis, pyrazinamide-resistant technology, which is applied in the biological field to achieve the effects of high sensitivity, shortened time, and short detection time

Active Publication Date: 2016-04-06
转换医学生物科技(云南)有限公司
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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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  • Primer set for detection of Mycobacterium tuberculosis and its resistance to pyrazinamide
  • Primer set for detection of Mycobacterium tuberculosis and its resistance to pyrazinamide
  • Primer set for detection of Mycobacterium tuberculosis and its resistance to pyrazinamide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The pyrazinamide-sensitive strain Mycobacterium tuberculosis standard strain M.tuberculosisH37Ra (American Culture Collection Center No.: ATCC25177) was used as the pyrazinamide drug-sensitive control, and the standard pyrazinamide-resistant strain BCG (American Culture Collection Center No.: ATCC19274) as pyrazinamide drug resistance control, the assay 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 test whether this method can distinguish the standard pyrazinamide Zinamide-resistant and susceptible strains.

[0065] Specific steps are as follows:

[0066] 1. Lysis of Mycobacterium tuberculosis and extraction of its DNA:

[0067] (1) From the L-J medium cultured with the standard strains of Mycobacterium tuberculosis M.tuberculosisH37Ra and BCG, scrape a ring of bacteria with an inoculation loop, dissolve in 2mL NaOH soluti...

Embodiment 2

[0096] In this embodiment, 3 parts of clinical sputum (No. 218, 271, and No. 8333) of tuberculosis patients diagnosed as tuberculosis patients collected from Wuhan Tuberculosis Prevention and Control Institute are used as test samples to test whether this method can be suitable 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:

[0097] 1. Cracking and extracting the DNA of Mycobacterium tuberculosis in sputum;

[0098] (1) Add 2mL NaOH solution to a certain amount of sputum (1-5mL), mix well, and incubate at 37°C for 20min.

[0099] (2) Centrifuge for 10 minutes, remove the supernatant and keep the precipitate. Wash with PBS, repeat the wash 2 times, remove the supernatant and save the precipitate for detection.

[0100] (3) DNA extraction: add purified water of DNA extraction solution to the above-mentioned processed specimen. Boiling...

Embodiment 3

[0121] 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. In this embodiment, the pyrazinamide-sensitive strain Mycobacterium tuberculosis standard strain M.tuberculosisH37Ra (American Culture Collection Center No.: ATCC25177) is used as a pyrazinamide drug-sensitive control, and the standard pyrazinamide-resistant strain BCG (American Culture Collection Center) Center No.: ATCC19274) as pyrazinamide resistance control, two 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.

[0122] Specific steps are as follows:

[0123] 1. Using the same method as in Example 1 to lyse Mycobacterium tuberculosis standard s...

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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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Patent Type & Authority Patents(China)
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 转换医学生物科技(云南)有限公司
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