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Genotype detection primer group and kit for mycobacterium tuberculosis

A technology for Mycobacterium tuberculosis and genotyping, applied in microorganism-based methods, microorganisms, recombinant DNA technology, etc., and can solve problems such as differences in discrimination ability, low typing index, poor stability and repeatability

Inactive Publication Date: 2014-06-04
中华人民共和国吉林出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the sites and primer sets currently used in the MLVA detection of Mycobacterium tuberculosis still have problems of poor stability and repeatability, and the typing index is not high
Moreover, due to the regional characteristics of the prevalence of Mycobacterium tuberculosis, different VNTR sites and primer combinations show differences in the discrimination ability when studying strains from different regions or different sources.

Method used

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  • Genotype detection primer group and kit for mycobacterium tuberculosis
  • Genotype detection primer group and kit for mycobacterium tuberculosis
  • Genotype detection primer group and kit for mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Extraction of Mycobacterium tuberculosis Genomic DNA

[0087] Take 48 strains of Mycobacterium tuberculosis to be tested, pick 2-3 colonies for each strain, put them into centrifuge tubes respectively, add 200 μL TE buffer solution and add SDS to make the final concentration 1%, and store at 85°C-90°C After cooking at ℃ for 10 minutes, the genomic DNA of Mycobacterium tuberculosis was extracted according to the bacterial DNA extraction kit of Takara Company. The obtained genomic DNA was stored at -20°C before testing.

Embodiment 2

[0088] Example 2: PCR amplification of different VNTR sites of the strains to be tested

[0089] With 22 pairs of primers in the Mycobacterium tuberculosis genotyping detection primer set provided by the present invention, PCR amplification is carried out to the VNTR sites in 48 strains to be tested respectively:

[0090] The amplification system is:

[0091]

[0092] The PCR reaction conditions for the sites MIRU4, MIRU10, MIRU31, MIRU39, MIRU26, MIRU2, MIRU24, MIRU16, MIRU40, MIRU20, MIRU23 and MIRU27 are:

[0093]

[0094] The PCR reaction conditions for sites ETR A, QUB-18, ETR B, ETR C, QUB-11a and QUB-26 are:

[0095]

[0096] The PCR reaction conditions for sites QUB-3232, QUB-3336, QUB-1895, QUB-4156 and QUB-2163 are:

[0097]

[0098] The PCR reaction conditions for sites Mtub34, Mtub29, Mtub39, Mtub30, Mtub21 and Mtub4 are:

[0099]

[0100] After PCR amplification, the PCR products of 22 VNTR sites in 48 strains were obtained.

Embodiment 3

[0101] Embodiment 3: Electrophoresis and fragment copy number identification of PCR products

[0102] The PCR products of each VNTR site in the 48 strains were detected by capillary electrophoresis to determine the fragment size of the PCR products.

[0103] The conditions of capillary electrophoresis are as follows:

[0104] The sample volume is 5 μL, voltage: 100V-240V, 50 / 60Hz; working temperature: 10°C-30°C; working humidity: 10%-75%. The cartridges used are QX DNA Screening Cartridge and QX DNA High Resolution Cartridge respectively.

[0105] Running program: Use the program that comes with the system software, the detection time is 450s, and the sampling time is 10s.

[0106] According to the length of the product, the copy number of the VNTR locus is converted. If the copy number of each VNTR locus is consistent, it is judged as the same genotype;

[0107] Wherein, the copy number of the VNTR site=PCR product size / length of each copy of the VNTR site. The calculatio...

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Abstract

The invention relates to the field of genotypes, and in particular relates to a genotype detection primer group and a kit for mycobacterium tuberculosis. The primer group comprises 22 primer pairs for detecting 22 VNTR loci in a mycobacterium tuberculosis genome. The primer group can be used for detecting the diversity of the 22 VNTR loci of the mycobacterium tuberculosis, so that the aim of genotyping the mycobacterium tuberculosis can be fulfilled. Experimental results show that high resolving power and high specificity are ensured when the primer group is used for mycobacterium tuberculosis genotype detection, each locus is high in polymorphism, and the genotyping index can reach 0.903.

Description

technical field [0001] The invention relates to the field of genotyping, in particular to a primer set for genotyping detection of Mycobacterium tuberculosis and a kit thereof. Background technique [0002] Tuberculosis (TB), caused by Mycobacterium tuberculosis infection, is still one of the important infectious diseases that endanger human health. Mycobacterium tuberculosis, commonly known as Mycobacterium tuberculosis or Mycobacterium tuberculosis, is prone to variation in morphology, colony, virulence, immunogenicity and drug resistance. Moreover, because tuberculosis has the characteristics of latent infection, traditional epidemiological methods cannot reveal its transmission rules well. At present, the main method to study the molecular epidemiology of tuberculosis is genotyping technology, which can track pathogenic bacteria, distinguish whether tuberculosis is exogenous infection or recurrence of old disease, and identify cross-infection in the laboratory. Technol...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/32
CPCC12Q1/04C12Q1/686C12Q2531/113
Inventor 孟庆峰王伟利杨莉李海滨
Owner 中华人民共和国吉林出入境检验检疫局
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