SNP molecular markers and methods, primer compositions, kits and applications for the identification of mycobacteria

Abstract

The invention provides a SNP molecular marker, a method, a primer composition, a kit and an application for identifying mycobacteria, and relates to the technical field of bacterial species identification. The SNP molecular marker contains a total of 17 SNP sites, which are distributed in the Hsp65 gene, rrs gene and pncA gene. Mycobacterium endogenous, Mycobacterium fortuitum, Mycobacterium kansasii, Mycobacterium gastric, Mycobacterium marinum, Mycobacterium microti, Mycobacterium scrofula, Mycobacterium smegmatis, Mycobacterium suja, Abscessus Polymorphisms in Mycobacterium, Mycobacterium ulcerans, Mycobacterium xenopus, Mycobacterium gordonii and Mycobacterium simiensis. The above primer composition can amplify Hsp65 gene, rrs gene and pncA gene, so as to be used for subsequent analysis of SNP molecular markers and identification of strains.

Description

technical field

[0001] The invention relates to the technical field of bacterial species identification, in particular to a SNP molecular marker and method, primer composition, kit and application for identifying mycobacteria. Background technique

[0002] Tuberculosis is one of the most serious epidemic infectious diseases, which makes human public health face a huge challenge. It has a very high morbidity and mortality rate worldwide.

[0003] Traditional methods of 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 restricted to slow bacteria. Cultivation, but also involves subjective interpretation prone to error. At present, the common molecular biology method for bacterial species identification is mainly based on the determination of the base difference of the 16s rRNA in the inserted sequence. Even ...

Examples

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, as shown in Table 4.

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

[0089]

Embodiment 2

[0091] The primer compositions provided in Example 1 were used to identify 18 species of mycobacteria, including M. tuberculosis (ATCC 27294), M. bovis (ATCC 19210) and Tian who belong to the Mycobacterium tuberculosis complex Mycobacterium murine M. Microti (ATCC 19422); also Mycobacterium avium (ATCC 25291) belonging to non-tuberculous mycobacteria (MOTT) outside the Mycobacterium tuberculosis complex. All strains were kept in the Beijing Tuberculosis Clinical Data and Sample Repository of Beijing Chest Hospital Affiliated to Capital Medical University.

[0092] Using the genomic DNA of the tested strain as a template, the primer pair designed in Example 1 was used for PCR amplification.

[0093] PCR reaction: use the three sets of primer pairs in Table 4, and use a conventional PCR reaction system. The reaction system is as follows: the total volume is 20 μl, including 2×Premix Taq (Code No.: R004A, Takara Company, Premix is ​​made of DNA Polymerase, Buffer , 2 times the c...

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) to extract DNA through pretreatment;

[0100] 2. PCR reaction: use the primer pairs of the three groups 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 No.: R004A, Takara Company, Premix is ​​produced by DNAPolymerase , Buffer, dNTP Mixture) 10 μl, 10 μM upstream and downstream primers each 1 μl (final concentration is 0.5 μM), 1 μl DNA template, supplemented with 7 μl of double distilled water to 20 μl system.

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

technical field

[0001] The invention relates to the technical field of bacterial species identification, in particular to a SNP molecular marker and method, primer composition, kit and application for identifying mycobacteria. Background technique

[0002] Tuberculosis is one of the most serious epidemic infectious diseases, which makes human public health face a huge challenge. It has a very high morbidity and mortality rate worldwide.

[0003] Traditional methods of 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 restricted to slow bacteria. Cultivation, but also involves subjective interpretation prone to error. At present, the common molecular biology method for bacterial species identification is mainly based on the determination of the base difference of the 16s rRNA in the inserted sequence. Even ...

Examples

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, as shown in Table 4.

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

[0089]

Embodiment 2

[0091] The primer compositions provided in Example 1 were used to identify 18 species of mycobacteria, including M. tuberculosis (ATCC 27294), M. bovis (ATCC 19210) and Tian who belong to the Mycobacterium tuberculosis complex Mycobacterium murine M. Microti (ATCC 19422); also Mycobacterium avium (ATCC 25291) belonging to non-tuberculous mycobacteria (MOTT) outside the Mycobacterium tuberculosis complex. All strains were kept in the Beijing Tuberculosis Clinical Data and Sample Repository of Beijing Chest Hospital Affiliated to Capital Medical University.

[0092] Using the genomic DNA of the tested strain as a template, the primer pair designed in Example 1 was used for PCR amplification.

[0093] PCR reaction: use the three sets of primer pairs in Table 4, and use a conventional PCR reaction system. The reaction system is as follows: the total volume is 20 μl, including 2×Premix Taq (Code No.: R004A, Takara Company, Premix is ​​made of DNA Polymerase, Buffer , 2 times the c...

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) to extract DNA through pretreatment;

[0100] 2. PCR reaction: use the primer pairs of the three groups 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 No.: R004A, Takara Company, Premix is ​​produced by DNAPolymerase , Buffer, dNTP Mixture) 10 μl, 10 μM upstream and downstream primers each 1 μl (final concentration is 0.5 μM), 1 μl DNA template, supplemented with 7 μl of double distilled water to 20 μl system.

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

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