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MNP (Mycobacterium tuberculosis) marker combination, primer pair combination, kit and application of MNP marker combination, primer pair combination and kit

A technology of mycobacterium tuberculosis and primer pairs, applied in the biological field, can solve problems such as lack of technology

Pending Publication Date: 2022-04-05
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in microorganisms, there is no report on MNP labeling, and there is a lack of corresponding technology

Method used

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  • MNP (Mycobacterium tuberculosis) marker combination, primer pair combination, kit and application of MNP marker combination, primer pair combination and kit
  • MNP (Mycobacterium tuberculosis) marker combination, primer pair combination, kit and application of MNP marker combination, primer pair combination and kit
  • MNP (Mycobacterium tuberculosis) marker combination, primer pair combination, kit and application of MNP marker combination, primer pair combination and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 The screening of Mycobacterium tuberculosis MNP marker combination and the design of multiple PCR amplification primers

[0032] S1, Screening of Mycobacterium tuberculosis MNP marker combinations

[0033] Based on the genome sequences of 501 different isolates of Mycobacterium tuberculosis published online, 17 MNP markers were obtained through sequence alignment. For species that do not have genome data on the Internet, the genome sequence information of representative races of the microbial species to be detected can also be obtained through high-throughput sequencing, where high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure the polymorphism of the selected markers, the genome sequences of at least 10 isolates are generally used as references.

[0034] The 17 MNP markers screened are shown in Table 1:

[0035] Table 1 The starting position of the MNP markers and detection primers on the reference genome

[0...

Embodiment 2

[0048] Performance evaluation and threshold formulation of MNP marker combination and kit for identifying Mycobacterium tuberculosis described in Example 2

[0049] In this example, the DNA of Mycobacterium tuberculosis was added into the Into human genomic DNA, prepare 1 copy / reaction, 10 copies / reaction, and 100 copies / reaction of M. tuberculosis mock samples. At the same time, an equal volume of sterile water was set as a blank control. In this embodiment, there are a total of 4 samples, and 3 replicate libraries are constructed for each sample every day, and continuously detected for 4 days, that is, 12 sets of sequencing data are obtained for each sample, as shown in Table 2. According to the number of sequencing fragments and the number of markers of Mycobacterium tuberculosis MNP markers detected in the blank control and Mycobacterium tuberculosis mock samples in 12 repeated experiments, the reproducibility, accuracy and sensitivity of the detection method were evaluat...

Embodiment 3

[0074] Example 3, Genetic variation detection between the same named Mycobacterium tuberculosis strains

[0075] In experimental research, the variation of strains with the same name will lead to incomparable and irreproducible experimental results. A total of 10 attenuated strains of Mycobacterium tuberculosis BCG provided by Huazhong Agricultural University, Wuhan Institute of Virology and Shihezi University were detected using the kit and MNP marker combination detection method, and the samples were named S1-S10 in turn, each The average sequencing coverage of the samples reached 2103 times, and all 17 MNP markers could be detected on average for each strain (Table 5). The fingerprints of the 10 strains were pairwise compared, and the results are shown in Table 6. The MNP fingerprints of the 3 copies of BCG kept in laboratories A and C were consistent, and among the 4 copies of laboratory B, 1 Partially marked major genotype differences existed in the batch (S-5) and the 9...

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Abstract

The invention discloses an MNP marker combination of mycobacterium tuberculosis, a primer pair combination, a kit and application of the MNP marker combination, the primer pair combination and the kit. The MNP marker combination comprises markers of MNP-1 to MNP-17 on an AL123456 genome, and the specific nucleotide sequences of the markers are shown as SEQ ID NO. 1 to SEQ ID NO. 17. The nucleotide sequence of the primer pair combination is as shown in SEQ ID NO. 18 to SEQ ID NO. 51. The MNP marker combination can specifically identify mycobacterium tuberculosis and finely distinguish different variants; the primers do not interfere with each other, integrate multiple amplification and sequencing technologies, can perform sequence analysis on all marker combinations of multiple samples at one time, and have the technical advantages of high throughput, multiple targets, high sensitivity, high accuracy and no culture; the method can be applied to fine identification, heritable variation detection, fingerprint database construction and the like of the mycobacterium tuberculosis of large-scale samples, and has important significance on monitoring, prevention and scientific research of the mycobacterium tuberculosis.

Description

technical field [0001] The embodiment of the present invention relates to the field of biotechnology, in particular to a MNP marker combination of Mycobacterium tuberculosis, a primer pair combination, a kit and applications thereof. Background technique [0002] Mycobacterium tuberculosis (Mycobacterium tuberculosis), commonly known as Mycobacterium tuberculosis. Mycobacterium tuberculosis can invade susceptible organisms through respiratory tract, digestive tract or skin lesions, causing tuberculosis in various tissues and organs. Mycobacterium tuberculosis can be inhaled through droplets or dust containing bacteria, so it is more common in pulmonary tuberculosis. Tuberculosis is a worldwide disease and one of the most public health concerns. Although in recent years, with the production of vaccines and early vaccination of children, the incidence of tuberculosis has shown a downward trend, but its harm to human health is still very serious. Rapid and accurate detection...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6858C12Q1/04C12N15/11
CPCY02A50/30
Inventor 肖华锋高利芬陈利红李甜甜周俊飞彭海李论方治伟万人静
Owner JIANGHAN UNIVERSITY
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