Detection primers for LAMP (loop-mediated isothermal amplification) of multi-drug resistant gene oqxAB, detection kit and detection method

A ring-mediated isothermal and multi-drug resistance technology, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of PCR false positive, long detection time, high detection cost, etc., and achieve specificity Strong, long solution time, high sensitivity effect

Inactive Publication Date: 2017-05-10
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems that PCR amplification is prone to false positives, high detection cost, complicated operation, and long detection time, LAMP is added to detect multi-drug resistance genes wxya blank, the present invention provides the use of loop-mediated isothermal amplification of multi-drug resistance genes wxya Primers

Method used

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  • Detection primers for LAMP (loop-mediated isothermal amplification) of multi-drug resistant gene oqxAB, detection kit and detection method
  • Detection primers for LAMP (loop-mediated isothermal amplification) of multi-drug resistant gene oqxAB, detection kit and detection method
  • Detection primers for LAMP (loop-mediated isothermal amplification) of multi-drug resistant gene oqxAB, detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation and detection of multiple drug resistance genes wxya Kit

[0039] (1) Design of detection primers

[0040] Since the length of the sequence is generally required to be ≤2000 bp when designing LAMP primers, the currently known wxya Most of the gene sequences are 5140 bp. Therefore, first of all, according to the known wxya The sequence of the gene, comparing the three strains of Escherichia coli, Salmonella and Klebsiella pneumoniae wxya Gene sequence, find out the conserved region, design a pair of specific inner primers FIP, BIP, a pair of specific outer primers F3, B3, and loop primers LF, LB, respectively, for multi-drug resistant genes wxya to test. During the experiment, several pairs of primers were designed, and the primers with the best effect were selected according to the test results. The base sequence is as follows:

[0041] Outer primer F3: 5'-TGCGCGCGGAGTATCC-3' (SEQ No.1)

[0042] Outer primer B3: 5'-GAACCTGCGCCTGATCC-3' (SEQ...

Embodiment 2

[0056] Example 2: Detection of multidrug resistance genes by loop-mediated isothermal amplification wxya detection method

[0057] (1) Extract the genomic DNA of the clinical strains to be tested

[0058] After culturing the clinical strains to be tested overnight, take 1 mL of the bacterial solution and centrifuge at 12,000 rpm for 2 min, and discard the supernatant completely. Take the precipitate and extract the total DNA of the bacterial genome with a bacterial genomic DNA extraction kit, and finally dissolve it in 50 μl sterilized double-distilled water, and store it at -20°C for future use.

[0059] (2) LAMP amplification

[0060] Using the kit in Example 1, set up a detection tube, a positive control tube and a negative control tube. Add 2 μl of the DNA sample to be tested in the test tube, and add 2 μl of the positive control tube containing wxya Escherichia coli genomic DNA (DNA concentration: 52 ng / μl), add 2 μl sterilized double distilled water to the negative c...

Embodiment 3

[0063] Example 3: wxya Sensitivity comparison between gene LAMP detection method and common PCR method

[0064] Take and contain wxya Genomic DNA of Escherichia coli (concentration: 52 ng / μl), diluted by 10 times, respectively 10 of the stock solution -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 times, 2 μl were used as templates for LAMP and PCR reactions. In the LAMP reaction tube and the PCR reaction tube, add the above doubly diluted containing wxya Genetic bacterial genomic DNA 2 μl.

[0065] Among them, the LAMP reaction tube was placed in a constant temperature water bath at 65 °C for 45-60 min. After the reaction, the color change was directly observed with the naked eye, and the result was verified by electrophoresis.

[0066] All primers in the PCR reaction are known according to NCBI wxya The conserved sequence of the gene was designed with Primer 5.0 software, and synthesized by a DNA synthesizer according to the following base sequence:

[0067] Upstream prime...

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Abstract

The invention relates to the technical field of bacterial gene detection, in particular to primers, shown as sequences 1-6 in the sequence table, for LAMP (loop-mediated isothermal amplification) of a multi-drug resistant gene oqxAB, a detection kit used for detecting the multi-drug resistant gene oqxAB with LAMP and containing the primers shown as the sequences 1-6 in the sequence table, as well as a detection method for detecting the multi-drug resistant gene oqxAB with LAMP. With adoption of the LAMP technology, the specificity is high, the sensitivity is higher than that with a PCR detection method, and the defects of long time, high workload, cross contamination, complicated operation and the like in the prior art are overcome; LAMP is rapid, low in cost and applicable to field rapid detection, and an operation method is simpler.

Description

technical field [0001] The invention relates to the technical field of bacterial gene detection, in particular to loop-mediated isothermal amplification of multiple drug-resistant genes wxya Primers for detection of multi-drug resistance genes by a loop-mediated isothermal amplification method wxya The kit also involves the detection of multidrug resistance genes by the loop-mediated isothermal amplification method wxya detection method. Background technique [0002] Plasmid-encoded multidrug resistance efflux pump wxya It was first discovered when studying the mechanism of olaquindox resistance. In 2003, Danish scientist Sorensen et al. first discovered the plasmid pOLA52 that mediates drug resistance to olaquindox in Escherichia coli isolated from pig feces samples. In 2004, Hansen et al. found the efflux of olaquindox resistance from the pOLA52 plasmid pump gene wxya . Because its drug resistance mechanism is very consistent with the active efflux pump character...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 李璐璐刘玉庆刘玲红胡明齐静张庆骆延波张印
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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