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High-throughput sequencing method for mycobacterium typing

A technology of mycobacteria and detection method, which is applied in the field of high-throughput sequencing of mycobacteria typing, can solve the problems that cannot meet the clinical needs, and achieve the effect of shortening the required time, reducing pain and high accuracy

Pending Publication Date: 2018-02-23
JIAXING YUNYING MEDICAL INSPECTION CO LTD
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AI Technical Summary

Problems solved by technology

Commonly used methods include PCR-RFLP, PCR-direct sequencing and DNA probe, etc. These methods have their own advantages and disadvantages, but still cannot meet the clinical needs. Therefore, it is urgent to establish a fast, simple and accurate method for the identification of mycobacterial species

Method used

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Embodiment Construction

[0026] A kind of high-throughput detection method of identification mycobacteria typing of the present invention, comprises the steps:

[0027] 1. Sample library preparation:

[0028] (1) Pipette 60 ul of purified DNA and 40 ul of End prepmix into a single PCR tube, mix by pipetting, centrifuge briefly, and perform end repair. The reaction conditions are 30°C and 30 min.

[0029] (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 end-repaired PCR product, beat and mix, and incubate in the greenhouse for 5 minutes.

[0030] (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 Rinse twice with % ethanol, and dry at room temperature for 10 minutes (with 2 minutes remaining, use a small grab to s...

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Abstract

The invention discloses a method for detecting mycobacterium typing. The method selects the most common genome information of mycobacterium tuberculosis and non-tuberculous mycobacteria at present, and combines the second-generation high-throughput sequencing technology to more comprehensively detect and identify the mycobacterium strain typing situation. Compared with PCR-RFLP and PCR-direct sequencing methods and a DNA probe identification method, the second-generation high-throughput sequencing technology has large throughput, short sequencing time, high accuracy, low cost and rich information, and accurate detection and identification of strains in a short period of time. With the help of high-throughput detection technology, the method can provide clinicians with more detailed straininformation of mycobacteria infected by patients, help doctors to make more accurate treatment plans, and reduce the treatment course of diseases, cost and adverse reaction.

Description

technical field [0001] The invention relates to a high-throughput sequencing method for typing mycobacteria. Background technique [0002] Mycobacteria are one of the most important pathogenic microbiota in human history. Among the more than 250 species of Mycobacterium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti and Mycobacterium africanum are also called Mycobacterium tuberculosis complex because of their close biological characteristics. Mycobacteria in the genus Mycobacterium except Mycobacterium tuberculosis complex and Mycobacterium leprae (which is rare in clinical practice) are collectively referred to as non-tuberculous mycobacteria, such as Mycobacterium avium and Mycobacterium intracellulare. [0003] The main pathogens of human tuberculosis are strains of Mycobacterium tuberculosis complex such as Mycobacterium tuberculosis and Mycobacterium bovis. Mycobacterium tuberculosis is the pathogenic bacterium that causes tuberculosis, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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