Oligonucleotide primer for detecting common pathogenic bacteria by adopting fluorescent quantitation PCR (Rich Client Platform) technology, method thereof for detecting common pathogenic bacteria and application thereof

An oligonucleotide and fluorescent quantitative technology, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc. Infection and other problems, to achieve the effect of high-efficiency application range

Inactive Publication Date: 2010-12-29
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Conventional PCR can realize the rapid detection of microorganisms, especially pathogenic bacteria, but it cannot quantify the pathogenic bacteria to be detected, and only one kind of pathogenic bacteria can be detected in one experiment, and the efficiency is low; quantitative PCR can be carried out for the pathogenic bacteria to be detected Quantitative detection, but one experiment can only detect one pathogenic bacteria to be tested, and cannot ac

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Taking the simultaneous detection of Salmonella and Listeria monocytogenes in water as an example

[0081] 1. Carry out the design of Salmonella and Listeria monocytogenes specific oligonucleotide primers: primer design is a crucial part in the PCR technology, especially in the multiple pathogen detection method and application of the present invention In the system, it is the most important factor in the whole system; the steps of specific oligonucleotide primer design are as follows:

[0082] ⑴Log in to GenBank (http: / / ncbi.nlm.nih.gov / entrez), search for the target gene fimA sequence of Salmonella and the target gene hly sequence of Listeria monocytogenes in the nucleic acid database, and screen out after checking one by one Sequences with good sequencing quality;

[0083] (2) Use Primer Premier 5.0 software to design specific oligonucleotide primers for the target gene sequence;

[0084] (3) Submit the designed candidate sequences to GenBanK for BLAST co...

Embodiment 2

[0095] Example 2 Taking the simultaneous detection of Shigella and Campylobacter jejuni in food as an example

[0096] Ⅰ. Design Shigella and Campylobacter jejuni-specific oligonucleotide primers: the steps of oligonucleotide primer design are as follows:

[0097] (1) Log in to GenBank (http: / / ncbi.nlm.nih.gov / entrez), search for the ipaH sequence of the target gene of Shigella and the htp sequence of the target gene of Campylobacter jejuni in the nucleic acid database, and check the sequencing quality one by one better sequence;

[0098] (2) Use Primer Premier 5.0 software to design specific primers for the target gene sequence;

[0099] (3) Submit the designed candidate sequences to GenBanK for BLAST comparison, and select the one with the best specificity for synthesis. The primers for Shigella are Sf-ipa-1 (5'- CCGGGATAAAGTCAGAACTC-3') and Sf-ipa -2(5'-CTCCCGACACGCCATAGAAA-3'), the length of the PCR product of this pair of primers is 131bp; the primers of C. '- AGGCACGCCT...

Embodiment 3

[0110] Example 3 Taking the simultaneous detection of Enterobacter sakazakii and enterohaemorrhagic Escherichia coli O157 in feces as an example

[0111] Ⅰ. Design of specific primers for Enterobacter sakazakii and E. coli O157: The steps of primer design are as follows:

[0112] ⑴Log in to GenBank (http: / / ncbi.nlm.nih.gov / entrez), search the target gene ompA sequence of Enterobacter sakazakii and the target gene eae sequence of enterohaemorrhagic Escherichia coli O157 in the nucleic acid database, check and screen Generate sequences with better sequencing quality;

[0113] (2) Use Primer Premier 5.0 software to design specific primers for the target gene sequence;

[0114] (3) Submit the designed candidate sequences to GenBank for BLAST comparison, and select the one with the best specificity for synthesis. The primers for Enterobacter sakazakii are Es-ompA-1 (5'- GCTGAGCGTAGGTGTTTCCT-3') and Es-ompA -2 (5'-CAGGGTGAAGTGCTTGGTCT-3' ), the PCR product of this pair of primers ...

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Abstract

The invention discloses an oligonucleotide primer for detecting common pathogenic bacteria by adopting a fluorescent quantitation PCR (Rich Client Platform) technology, a method thereof for detecting common pathogenic bacteria and the application thereof. The method comprises the following steps of: providing 10 pairs of specific oligonucleotide primer sequences at annealing temperature of 50-60 DEG C without differing 5 DEG C; and simultaneously, quickly, accurately and effectively identifying and quantificationally detecting various pathogenic bacteria at the same time. A detection range comprises bacillus cereus, enterobacter sakazakii, vibrio parahaemolyticus, enterohemorrhagic escherichia coli O157, salmonella, Listeria monocytogenes, Shigella, campylobacter jejuni, pseudomonas aeruginosa, klebsiella pneumoniae, and the like. The invention also can be used for the fields of disease diagnosis, environmental monitoring, water-quality and food supervision and detection, food poisoning pathogenicbacteria detection, bacteriological classification, epidemiological investigation, biological agent detection, and the like, is convenient, quick, accurate and effective and has wide application range.

Description

technical field [0001] The invention relates to the technical field of pathogenic bacteria detection, in particular to a convenient, fast, accurate, efficient and wide application oligonucleotide primer for detecting common pathogenic bacteria using fluorescence quantitative PCR technology and detecting common pathogenic bacteria Methods and uses of bacteria. Background technique [0002] As we all know, the detection of pathogenic bacteria has important applications in the fields of disease diagnosis, environmental monitoring, food poisoning detection, import and export food quarantine, scientific research and other fields. Traditional pathogenic bacteria testing methods still remain at the level of isolation and culture, morphological observation, biochemical identification, and serological typing, which not only takes a long time, but also cannot effectively detect pathogenic bacteria that are difficult to culture, and can only be used for limited species one by one. det...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12Q1/06C12N15/11
CPCY02A50/30
Inventor 李君文孙飞龙金敏邱志刚谌志强陈照立王新为
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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