Mycobacterium tuberculosis rifampicin and isoniazide drug-resistant mutation detection kit and method

A technology for tuberculosis branching and drug-resistant mutations, which is applied in the fields of biotechnology and molecular diagnosis, and can solve problems such as cross-contamination, long cycle time, and high cost

Inactive Publication Date: 2021-06-11
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the disadvantage is that the cycle is long, the cost is high, and cross-contamination is prone to occur

Method used

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  • Mycobacterium tuberculosis rifampicin and isoniazide drug-resistant mutation detection kit and method
  • Mycobacterium tuberculosis rifampicin and isoniazide drug-resistant mutation detection kit and method
  • Mycobacterium tuberculosis rifampicin and isoniazide drug-resistant mutation detection kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0193] Embodiment 1. Kit and detection method

[0194]The composition, packaging and quantity of the Mycobacterium tuberculosis rifampicin and isoniazid resistance mutation detection kit (take 24 servings / box as an example), as shown in Table 6:

[0195] Table 6 The composition, packaging and quantity of the kit

[0196]

[0197] The specific implementation steps are as follows:

[0198] 1. Sample processing and nucleic acid extraction (sample processing area)

[0199] It is recommended to use nucleic acid extraction or purification reagents produced by Sun Yat-Sen University Da’an Gene Co., Ltd. (Yue Sui Ji Bei No. 20181263); please follow the instructions of the kit for specific operation steps. The internal standard solution in this kit needs to participate in the extraction process of each sample. If the extraction kit does not specify the method of using the internal standard, please add the internal standard solution to each sample according to the ratio of sample v...

Embodiment 2

[0217] Embodiment 2: Specific detection

[0218] Use this kit to detect 10 copies of isoniazid and rifampicin-sensitive national reference materials for Mycobacterium tuberculosis rifampicin and isoniazid resistance gene detection reagents (purchased from China Institute for Food and Drug Control) The products, numbered N1-N10, were tested for specificity using the method of Example 1, and the results are shown in Table 8.

[0219] Table 8 Test results of sensitive national reference products

[0220] Numbering Test results N1 mutation negative N2 mutation negative N3 mutation negative N4 mutation negative N5 mutation negative N6 mutation negative N7 mutation negative N8 mutation negative N9 mutation negative N10 mutation negative

Embodiment 3

[0221] Embodiment 3: Accuracy detection

[0222] Use this kit to detect 32 drug-resistant rifampicin and isoniazid-resistant samples of Mycobacterium tuberculosis rifampicin and isoniazid-resistant gene detection reagents (purchased from China National Institute for Food and Drug Control) National reference products for genetic testing reagents. For rifampicin resistance sites, there are 531 codon mutations, 516 codon mutations, 526 codon mutations, 516 codon mutations, 522 codon mutations, 510-512 codon deletions, 513 codon mutations, and 533 codon mutations Mutation, deletion of codons 517-518, mutations of codons -15 and -8 of the inhA gene, mutations of bases -6 and -12 of the ahpC gene, and codon 315 of the katG gene for the isoniazid resistance locus mutation. The method of Example 1 was adopted for detection, and the results are shown in Table 9.

[0223] Table 9 Test results of drug-resistant national reference products

[0224]

[0225]

[0226] Negative con...

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Abstract

The invention provides a mycobacterium tuberculosis rifampicin and isoniazide drug-resistant mutation detection kit and method, and particularly discloses a method and kit for detecting mycobacterium tuberculosis rifampicin and isoniazide drug-resistant mutation based on a fluorescent PCR melting curve method. The method and kit can be used for rapidly and qualitatively detecting drug-resistant mutations of drug-resistant determining regions of rifampicin drug-resistant genes rpoB and isoniazide drug-resistant genes katG, inhA and ahpC in a positive sputum culture sample of a mycobacterium tuberculosis complex of a tuberculosis patient in vitro.

Description

technical field [0001] The invention belongs to the field of biotechnology and molecular diagnosis. Specifically, the invention relates to a detection kit and a method for the detection of rifampicin and isoniazid resistance mutations of Mycobacterium tuberculosis. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB). Rifampicin (RIF) is a semi-synthetic derivative of rifamycin and is currently the most important first-line anti-MTB drug. Bactericidal effect. Studies have shown that more than 95% of rifampicin resistance is caused by mutations in the rifampicin resistance determining region (RRDR) of 81 bp (coding codon 507-533) of the rpoB gene including point Caused by mutation or short insertion or deletion mutation, the enzymatic activity of the DNA-dependent RNA polymerase β subunit is changed, and rifampicin cannot combine with the bacterial RNA polymerase β subunit to show drug resistance. The rpoB ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/32
CPCC12Q1/689C12Q1/686C12Q2563/107C12Q2527/107
Inventor 蒋析文朱小亚徐小解侯广山
Owner DAAN GENE CO LTD
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