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
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[0086] Example 1
[0087] This embodiment provides a primer composition for identifying mycobacteria, including primer pair Myco1, primer pair Myco2, and primer pair Myco3, as shown in Table 4.
[0088] Table 4 Primer pair Myco1, primer pair Myco2 and primer pair Myco3
[0089]
Example Embodiment
[0090] Example 2
[0091] The primer composition provided in Example 1 was used to identify 18 kinds of mycobacteria, including Mycobacterium tuberculosis M. tuberculosis (ATCC 27294) belonging to the Mycobacterium tuberculosis complex, and Mycobacterium bovis (ATCC19210) Wada Mycobacterium murine M. Microti (ATCC 19422); also includes Mycobacterium avium (ATCC 25291) that belongs to non-tuberculous mycobacteria (MOTT) outside of the Mycobacterium tuberculosis complex. All strains are strains preserved in the Beijing Tuberculosis Clinical Data and Sample Resource Bank of Beijing Chest Hospital, Capital Medical University.
[0092] Using the genomic DNA of the test 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, using a conventional PCR reaction system, the reaction system is as follows: the total volume is 20μl, including 2×Premix Taq (Code No.: R004A, Takara company, Pr...
Example Embodiment
[0097] Example 3
[0098] This embodiment provides a method for identifying mycobacteria, which includes the following steps:
[0099] 1. To collect clinical sputum samples, use QIAamp DNA Mini Kit (CAT.No.51304) to extract DNA through pretreatment;
[0100] 2. PCR reaction: using the three sets of primers in Table 4, using conventional PCR reaction system, the reaction system is as follows: the total volume is 20μl, including 2×Premix Taq (Code No.: R004A, Takara company, Premix is made of DNA Polymerase) , Buffer, dNTP Mixture) 10μl, 10μM upstream and downstream primers each 1μl (final concentration 0.5μM), DNA template 1μl, supplemented with double distilled water 7μl to 20μl 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. Using the same annealing temperature for the primers reduces the operation steps and saves the time for strain identification.
[0102] PCR results such as f...
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