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SNP molecular markerand method for identifying mycobacteria, primer composition, kit and application

A technique of primer composition and mycobacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of long time, easy false positives in picture results, low resolution, etc., to improve accuracy degree of effect

Active Publication Date: 2019-04-16
BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IS6110-RFLP is widely used and has high resolution, but this method is based on the cultivation of a large number of strains, which is expensive, takes a long time, and strains with a small number of copies of IS6110 are not easy to type; MIRU-VNTR does not require a large number of cultured strains, and the results are easy to read , but due to the different combinations of deoxynucleotide points, the resolution and clustering rate are also different; the Spoligotyping typing method takes less time and costs less, but the resolution is low, and the image results are prone to false positives

Method used

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  • SNP molecular markerand method for identifying mycobacteria, primer composition, kit and application
  • SNP molecular markerand method for identifying mycobacteria, primer composition, kit and application
  • SNP molecular markerand method for identifying mycobacteria, primer composition, kit and application

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Embodiment 1

[0087] This embodiment provides a primer composition for identifying mycobacteria, including a primer pair Myco1, a primer pair Myco2 and a primer pair Myco3, specifically as shown in Table 4.

[0088] Table 4 primer pair Myco1, primer pair Myco2 and primer pair Myco3

[0089]

Embodiment 2

[0091] Using the primer composition provided in Example 1 to identify 18 kinds of mycobacteria, including Mycobacterium tuberculosis M.tuberculosis (ATCC 27294) belonging to the Mycobacterium tuberculosis complex, Mycobacterium bovis M.Bovis (ATCC19210) and Tian Mycobacterium murine M. Microti (ATCC 19422); also includes Mycobacterium avium M. avium (ATCC 25291 ), which belongs to nontuberculous mycobacteria (MOTT) outside the Mycobacterium tuberculosis complex. All strains were strains preserved in the Beijing Tuberculosis Clinical Data and Sample Resource Bank of Beijing Chest Hospital Affiliated to Capital Medical University.

[0092] With the genomic DNA of test bacterial strain as template, adopt the primer pair that embodiment 1 designs to carry out PCR amplification.

[0093] PCR reaction: use the three sets of primer pairs in Table 4, and adopt a conventional PCR reaction system. The reaction system is as follows: the total volume is 20 μl, including 2×Premix Taq (Code...

Embodiment 3

[0098] The present embodiment provides a method for identifying mycobacteria, comprising the steps of:

[0099] 1. For the collection of clinical sputum samples, use the QIAamp DNA Mini Kit (CAT.No.51304) kit to extract DNA through pretreatment;

[0100] 2.PCR reaction: use the primer pair of three groups in table 4, adopt conventional PCR reaction system, the reaction system is as follows: the total volume is 20 μ l, including 2×Premix Taq (Code No.: R004A, Takara company, Premix is ​​made by DNAPolymerase , Buffer, dNTP Mixture) 10 μl, 10 μM upstream and downstream primers 1 μl (final concentration 0.5 μM), DNA template 1 μl, double distilled water 7 μl to 20 μl system.

[0101] Reaction conditions: 94°C for 5min, 30 cycles: 94°C for 45sec, 60°C for 45sec, 72°C for 50sec, and finally extension at 72°C for 7min. The same annealing temperature is used for the primers, which reduces the operation steps and saves the time for strain identification.

[0102] PCR results such as...

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Abstract

The invention provides an SNP molecular marker and method for identifying mycobacteria, a primer composition, a kit and application, and relates to the technical field of strain identification. The SNP molecular marker comprises seventeen SNP loca distributed in an Hsp65 gene, an rrs gene and a pncA gene, and when marked in mycobacterium tuberculosis, mycobacterium bovis, Africa mycobacterium, mycobacterium avium, intracellular mycobacterium, mycobacterium fortuitum, mycobacterium kansasii, mycobacterium gastri, mycobacterium marinum,volemycobacterium, mycobacterium scrofulaceum, mycobacteriumsmegmatis, suga mycobacterium, mycobacterium abscessus, mycobacterium ulcerans, toad mycobacterium, mycobacterium gordonae and ape mycobacterium, the SNP molecular marker has polymorphism. The primercomposition can amplify the Hsp65 gene, therrs gene and thepncA gene to be used for analyzing of the SNP molecular marker and strain identification.

Description

technical field [0001] The invention relates to the technical field of bacterial species identification, in particular to a SNP molecular marker and method for identifying mycobacteria, a primer composition, a kit and an application. Background technique [0002] Tuberculosis is one of the most serious epidemic infectious diseases, which poses a huge challenge to human public health. It has a high morbidity and mortality rate worldwide. [0003] Traditional methods for identifying Mycobacterium tuberculosis species are based on phenotypic characteristics such as colony morphology, oxygen preference, niacin accumulation, nitrate reductase activity, and growth kinetics, all of which are limited to slow bacteria. Cultivation, and it involves subjective interpretation and is easy to make mistakes. The current common method of molecular biology for bacterial species identification is mainly based on the determination of the base difference of the inserted sequence 16s rRNA. Eve...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/32
CPCC12Q1/689C12Q2600/156
Inventor 孙照刚段慧娟孔成成曹廷明
Owner BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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