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High-throughput detection method of genotypic drug-resistant locus of mycobacterium tuberculosis

A technology for the detection of Mycobacterium tuberculosis, which is applied in the field of high-throughput detection of drug-resistant loci of Mycobacterium tuberculosis genes, can solve the problems of limited number of gene loci, false positives, and influence on drug efficacy, so as to reduce pain and improve accuracy The effect of increasing and shortening the required time

Pending Publication Date: 2018-02-27
JIAXING YUNYING MEDICAL INSPECTION CO LTD
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Problems solved by technology

There are also limitations in the number of detected gene loci, and it is impossible to distinguish silent mutations of important genes, which leads to the disadvantage of false positive results and affects the efficacy of drugs

Method used

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  • High-throughput detection method of genotypic drug-resistant locus of mycobacterium tuberculosis
  • High-throughput detection method of genotypic drug-resistant locus of mycobacterium tuberculosis

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Embodiment Construction

[0031] A kind of detection method of Mycobacterium tuberculosis drug resistance site of the present invention, comprises the steps:

[0032] 1. Sample library preparation:

[0033] (1) Ultrasound fragmentation: the initial amount is 3ug, add nuclease-free water to 100ul and dilute to 30ng / ul. SCIENTZ08-Ⅲ cup-type ultrasonic cell pulverizer was used for ultrasonic fragmentation, and the setting parameters were: power 70%, interrupt for 3s, stop for 1s, and cycle for 30-60min.

[0034] (2) Take out the magnetic beads half an hour in advance to return to room temperature, shake and mix, take 180ul of magnetic beads and add them to the PCR product interrupted by ultrasound, beat and mix, and incubate in the greenhouse for 5 minutes.

[0035](3) Put the PCR single tube on the magnetic stand, let it stand for 5 minutes, remove the supernatant, keep the PCR tube on the magnetic stand, add 200ul of 80% ethanol (newly prepared) to rinse, let stand for 30s, remove the supernatant, 80 ...

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Abstract

The invention discloses a high-throughput detection method of a genotypic drug-resistant locus of mycobacterium tuberculosis. In the method, the drug-resistant locus of an antituberculous drug of themycobacterium tuberculosis can be detected more comprehensively, more effectively and more quickly by utilizing a mutation database of used drug-resistant mycobacterium tuberculosis and combining a second generation high-throughput sequencing technology. According to the method, the difficulty that a false positive result is obtained as the silent mutation of important genes cannot be distinguished due to the limit to the number of detection gene loca in other detection methods of the drug-resistant locus is overcome; under the help of a high-throughput detection technology, more comprehensivediagnosis information can be provided for treating the drug-resistant mycobacterium tuberculosis better, so that the curative effect is improved, and the use of low-efficiency drugs is reduced.

Description

technical field [0001] The invention relates to a high-throughput detection method for drug-resistant sites of Mycobacterium tuberculosis genes. Background technique [0002] Mycobacterium tuberculosis is a pathogenic bacterium that causes human tuberculosis. It is a typical intracellular pathogen. It has acquired a series of virulence components through evolution, and suppressed the host's immune response through various mechanisms to escape immune surveillance, leading to the occurrence of tuberculosis. . Tuberculosis is still an important infectious disease, which has caused great difficulties in the effective prevention and treatment of tuberculosis. According to the WHO, about 8 million new cases occur each year and at least 3 million people die from the disease. With the continuous development of vaccination, anti-tuberculosis drugs and the improvement of sanitary living conditions, the morbidity and mortality of tuberculosis once dropped sharply. However, in recent...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/04C12R1/32
CPCC12Q1/6869C12Q2535/122C12Q2565/519C12Q2531/113
Inventor 张道允巩子英叶建伟王伟
Owner JIAXING YUNYING MEDICAL INSPECTION CO LTD
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