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Method for detecting escherichia coli fluoroquinolone-resisting gyrA/parC gene point mutation

A technology of Escherichia coli and quinolones, which is applied in the point mutation of parC gene to detect the fluoroquinolone resistance of Escherichia coli gyrA, and achieves the effects of low cost, favorable popularization and favorable method.

Inactive Publication Date: 2015-05-13
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods are sensitive, accurate and specific for detecting point mutations, there are still some shortcomings in actual detection.

Method used

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  • Method for detecting escherichia coli fluoroquinolone-resisting gyrA/parC gene point mutation
  • Method for detecting escherichia coli fluoroquinolone-resisting gyrA/parC gene point mutation
  • Method for detecting escherichia coli fluoroquinolone-resisting gyrA/parC gene point mutation

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: Escherichia coli to be tested and standard bacterial strains E. coli Genomic DNA Extraction of ATCC25922

[0033] Use conventional bacterial genomic DNA extraction methods, or bacterial genomic DNA extraction commercial kits (product number: SK8225, Sangon Biotech) to extract the genomic DNA of Escherichia coli to be tested and the standard strain Escherichia coli ATCC25922, and the concentration of the final DNA sample is about 150ng / μL.

Embodiment 2

[0034] Example 2: Design of specific primer pairs for two single-plex PCR amplifications

[0035] Using the primer design software primer premier 6, the wild-type standard strain E. coli ATCC25922 gyrA gene and parC The gene is used as a template to design two primers for single-plex PCR amplification, and the designed amplification includes gyrA (Ser codon 83 and Asp codon 87) nucleotide sequences including pairs of primers, and parC Multiple pairs of primers including the nucleotide sequence of (Ser codon 80 and Glu codon 84), after verification of each pair of primers, the specific PCR primer pair was obtained gyrA-FR (The upstream primer is the single-stranded DNA shown in SEQ ID No.1, and the downstream primer is the single-stranded DNA shown in SEQ ID No.2) and parC-FR (The upstream primer is the single-stranded DNA shown in SEQ ID No.3, and the downstream primer is the single-stranded DNA shown in SEQ ID No.4).

[0036] The primers were synthesized by a profe...

Embodiment 3

[0037] Example 3: Design of dual fluorescent-labeled probes gyrP and parP

[0038] Use the probe design software Xpression primer 3.1 to design the nucleotide sequences of the dual fluorescently labeled probes gyrP (shown in SEQ ID No.5) and parP (shown in SEQ ID No.6), and entrust the designed probes to Modified and synthesized by a professional company, purified by HPLC.

[0039] The specific design of the probe is: connect the HEX fluorescent group at the 5' end of the fluorescent labeling probe gyrP, connect the BHQ2 fluorescence quenching gene at the 3' end; connect the FAM fluorescent group at the 5' end of the fluorescent labeling probe ParP, and connect the fluorescent group at the 3' end. The 'end is connected to the BHQ2 fluorescence quencher gene; the probe gyrP includes 25 nucleotides, covering the gyrA Gene encoding nucleotide sequence from Gly 81 to Ile 89 for detection gyrA (Ser codon 83 and Asp codon 87) base point mutation; probe ParP Consists of 29 nucleo...

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Abstract

The invention relates to a method for detecting escherichia coli fluoroquinolone-resisting gyrA / parC gene point mutation, and belongs to the technical field of gene mutation detection. The method comprises the following steps: (1) extracting Escherichia coli genome DNA; (2) by taking the target gene sequence of E.coli ATCC25922 as a template, respectively designing and synthesizing fluorescence labeling probes gyrP and parP covering gyrA (Der codon83 and Asp codon87), and primers; (3) drawing a PCR amplification and probe melting curve of a strain DNA sample to be detected, adding DNA polymerase which is in thermal stability and is lack of 5' nuclease activity to prevent the probes from hydrolysis, and taking E.coli ATCC25922 as control; (4) judging whether point mutation happens or not. Due to adoption of the fluorescent probes, the method is high in specificity, and the detection can be completed within 2 hours.

Description

technical field [0001] A method for detecting Escherichia coli resistance to fluoroquinolones gyrA , parC Gene point mutation method. Background technique [0002] Fluoroquinolone antibacterial drugs are a class of synthetic broad-spectrum antibacterial drugs. The main target in Escherichia coli is DNA gyrase and also acts on topoisomerase IV. Inhibition of any of these enzymes will inhibit bacterial growth and lead to bacterial death. [0003] With the widespread use of fluoroquinolones, bacteria rapidly develop drug resistance. Studies have shown that there are four main mechanisms of bacterial resistance to fluoroquinolones: 1. The target enzyme or target site of fluoroquinolones is changed; concentration reduction; 3, plasmid-mediated drug resistance; 4, modifying enzyme gene ( aac(6')Ib-cr ) mutations to modify fluoroquinolones to reduce the susceptibility of bacteria to drugs. Among them, because gyrA , parC Mutations at specific sites in the gene cause ami...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/19
CPCC12Q1/686
Inventor 王红宁邓春朋张安云雷昌伟杨鑫徐昌文管仲斌刘必慧张冬冬
Owner SICHUAN UNIV
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