MNP (Mycoplasma Pneumoniae) marker combination, primer pair combination, kit and application of MNP marker combination, primer pair combination and kit
A technology of mycoplasma pneumoniae and primer pairs, applied in the biological field, can solve the problems of lack of technology and detection standards
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Embodiment 1
[0038] Example 1 The screening of Mycoplasma pneumoniae MNP marker combination and the design of multiple PCR amplification primers
[0039] S1. Screening of Mycoplasma pneumoniae MNP markers
[0040] Based on the complete or partial genome sequences of 771 different isolates of Mycoplasma pneumoniae published online, 14 MNP markers were obtained through sequence alignment. For species that do not have genomic 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 genetically representative isolates are generally used as references.
[0041] The 14 MNP markers screened are shown in Table 1:
[0042] The MNP mark described in Table 1 and the starting position of the detection pr...
Embodiment 2
[0054] Threshold setting and performance evaluation of MNP markers and primers for identifying Mycoplasma pneumoniae described in Example 2
[0055] In this example, the Mycoplasma pneumoniae nucleotide standard with known copy number was added to human genomic DNA to prepare mock samples of Mycoplasma pneumoniae with 1 copy / reaction, 10 copies / reaction and 100 copies / reaction. At the same time, an equal volume of sterile water was set as a blank control. A total of 4 samples were constructed, and 3 replicate libraries were constructed per day for each sample, and were tested continuously for 4 days, that is, 12 sets of sequencing data were obtained for each sample, as shown in Table 2. According to the number of sequenced fragments and markers of MNP markers detected in the blank control and Mycoplasma pneumoniae nucleotide standards in 12 repeated experiments, the thresholds for contamination of the quality control system and detection of target pathogens were established, a...
Embodiment 3
[0080] Embodiment 3, detection of genetic variation between Mycoplasma pneumoniae bacterial strains
[0081] Using the kit and the MNP marker combination detection method to detect the collected 6 Mycoplasma pneumoniae strains, the samples were named S1-S6 in sequence, and the average sequencing coverage of each sample reached 1530 times, and each strain could All 14 MNP markers were detected (Table 5). The fingerprints of the 6 bacterial strains were compared in pairs, and the results are shown in Table 5. There were 1 (S-2) and 5 Mycoplasma pneumoniae detected together with the same batch. There were some major genotype differences of the markers (Table 5), there is variation among strains, which may belong to different isolates.
[0082] The application of the kit to identify genetic variation among bacterial strains by detecting MNP markers can be used to ensure the genetic consistency of the same named Mycoplasma pneumoniae strains in different laboratories, thereby ensu...
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