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MNP marker site of klebsiella pneumoniae, primer composition, kit and application of MNP marker site

A Klebsiella pneumoniae and primer composition technology, applied in the biological field, can solve the problems of a large number of SNP markers, a small number, and insufficient capture of allelic diversity, and achieve accurate detection of genetic variation and high-accuracy detection , The effect of high sensitivity detection

Active Publication Date: 2022-08-02
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This technology allows us to detect different types of bacteria called Klebsiela Pneumonias (KPs) with very specific markers that are easy to creature on paper. It also has several other benefits such as efficient use of DNA samples from these microorganisms, precise measurements during polymerase chain reaction(PCR), ability to identify changes caused by environmental factors like temperature variations over time, and potential health hazards associated therewith.

Problems solved by technology

This patents discuss various techniques related to studying Klebsiela pneumonims caused by certain types of viruses like KPIVAX LCMV, which may lead to illnesses similar symptoms when transmitted between humans. These challengings require quicker and precise monitoring of these germs while minimizing resource usage and costs associated therewith. Current assay systems lack precision due to variations among other factors involved during testing procedures.

Method used

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  • MNP marker site of klebsiella pneumoniae, primer composition, kit and application of MNP marker site
  • MNP marker site of klebsiella pneumoniae, primer composition, kit and application of MNP marker site
  • MNP marker site of klebsiella pneumoniae, primer composition, kit and application of MNP marker site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Screening of Klebsiella pneumoniae MNP marker sites and design of multiplex PCR amplification primers

[0044] S1. Screening of Klebsiella pneumoniae MNP marker sites

[0045] Based on the complete or partial genome sequences of 529 different Klebsiella pneumoniae isolates published online, 9 MNP marker sites were obtained through sequence alignment. For species for which there is no genome data online, the genome sequence information of the representative race of the microbial species to be detected can also be obtained by high-throughput sequencing, where the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure the polymorphism of the selected marker, the genome sequences of at least 10 genetically representative isolates are generally used as a reference. The 9 MNP marker sites screened are shown in Table 1:

[0046] Table 1 - The MNP marker site and the starting position of the detection primer on the referen...

Embodiment 2

[0057] Embodiment 2, the detection of Klebsiella pneumoniae by MNP site and primer

[0058] 1. Detection of MNP markers

[0059] Klebsiella pneumoniae mock samples of 1 copy / reaction, 10 copies / reaction and 100 copies / reaction were prepared using DNA from a Klebsiella pneumoniae enumeration standard of known copy number added to human genomic DNA. At the same time, an equal volume of sterile water was set as a blank control. A total of 4 samples were collected, and 3 replicate libraries were constructed for each sample every day, and were continuously detected for 4 days, that is, 12 sets of sequencing data were obtained for each sample, as shown in Table 2. The detection process of MNP labeling is as follows: image 3 shown. According to the number of sequencing fragments and loci of Klebsiella pneumoniae MNP loci detected in blank control and Klebsiella pneumoniae mock samples in 12 repeated experiments, the reproducibility, accuracy, Sensitivity, setting thresholds for ...

Embodiment 3

[0083] Example 3. Detection of genetic variation among Klebsiella pneumoniae strains

[0084] Six Klebsiella pneumoniae strains provided by Hubei Provincial Center for Disease Control and Prevention were detected using the kit and MNP marker site detection method. The samples were named S1-S6 in sequence, and five of them were of the same strain Progeny strains propagated during the period. The average coverage fold of each MNP locus was 3103 times, and all 9 MNP markers could be detected in each strain (Table 5). The fingerprints of the 6 strains were compared in pairs, and the results are shown in Table 5. There are 1 (S-2) and 5 Klebsiella pneumoniae detected in the same batch. There are major genes in some loci. Type differences (Table 5), there is variation between strains, may belong to different isolates.

[0085] Table 5-6 Detection and Analysis of Klebsiella pneumoniae

[0086]

[0087] As can be seen from Table 5, the application of the described kit to identif...

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Abstract

The invention discloses MNP marker sites of klebsiella pneumoniae, a primer composition, a kit and application of the MNP marker sites, the primer composition and the kit, and the MNP marker sites are genome areas which are screened on a klebsiella pneumoniae genome, are distinguished from other species and have multiple nucleotide polymorphisms in the species, and comprise marker sites of MNP-1 to MNP-9; the primers are as shown in SEQ ID NO. 1 to SEQ ID NO. 18. The MNP marker site can be used for specifically identifying klebsiella pneumoniae and finely distinguishing different subtypes; the primers do not interfere with one another, integrate multiple amplification and sequencing technologies, can perform sequence analysis on all marker sites of multiple samples at a time, have the detection advantages of high throughput, multiple target spots, high sensitivity and culture-free detection, can be applied to identification and genetic variation detection of klebsiella pneumoniae of large-scale samples, and have wide application prospects. The method is of great significance to scientific research and degradation monitoring of klebsiella pneumoniae.

Description

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Claims

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Application Information

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Owner JIANGHAN UNIVERSITY
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