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Multiple PCR primers, kit and detection method for detecting polymyxin drug resistance genes

A detection kit and drug resistance gene technology, which can be used in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection. and specificity, high sensitivity and specificity

Inactive Publication Date: 2018-08-10
青岛智烨生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional PCR detection methods can only detect one resistance gene mcr-1, and cannot detect multiple drug resistance genes at the same time, and the process is cumbersome. Therefore, it is urgent to establish a set of rapid and effective detection methods for the entire mcr gene family. Detection method

Method used

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  • Multiple PCR primers, kit and detection method for detecting polymyxin drug resistance genes
  • Multiple PCR primers, kit and detection method for detecting polymyxin drug resistance genes
  • Multiple PCR primers, kit and detection method for detecting polymyxin drug resistance genes

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Effect test

Embodiment 1

[0024] Embodiment 1 multiplex PCR detection method

[0025] (1) Primer design

[0026] According to the reported literature, the DNA sequences of mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 were obtained from NCBI as the target genes. Use primer premier 5.0 software to design primers with different lengths of PCR products on different target genes to ensure that different drug resistance genes can be clearly distinguished in the end. Then use the Vector NTI software to compare and analyze the specificity of the designed primers and the homology with the target gene, so that the homology of each primer with the target gene is 100%, and the homology with other genes is maintained. Low, to ensure that the primers are universal within the target gene, but specific between genes, and finally determine the sequence of 5 pairs of identification primers (see Table 1), and the primers were synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0027] Identification primers used in the exp...

Embodiment 2

[0037] Specific detection between the embodiment 2 primers

[0038] Escherichia coli MG1655 containing mcr-1, mcr-2, mcr-3, mcr-4, mcr-5 genes were combined for single, double, triple, quadruple and quintuple strains, and then for all strain combinations For multiplex PCR amplification, the specificity between the primers was verified by observing the electrophoresis results.

[0039] By agarose gel electrophoresis results (such as figure 2 As shown), the bands of each target gene are relatively clear, and there are no non-specific bands, which shows that the multiplex PCR system established in this experiment shows good specificity and sensitivity to different strain combinations, and it is preliminarily determined that This reaction system can effectively screen the genes of the mcr gene family.

Embodiment 3

[0040] The detection and identification of embodiment 3 wild strains

[0041]Use the tested mcr negative and positive strains (see Table 2 for strain information, kept by the applicant) as templates to identify them by multiplex PCR, and compare them with the detection results obtained by conventional methods for these wild-type strains to determine Whether the multiplex PCR reaction system is suitable for the detection of wild-type strains from different sources.

[0042] Table 2 wild-type strain information

[0043]

[0044] Among all 156 strains to be tested, 77 strains were detected to have the mcr drug-resistant gene (results such as image 3 shown), and all drug-resistant gene-positive strains correspond to the correct size of drug-resistant gene fragments and a single band. This statistical result is consistent with the identification and detection results of these strains using traditional methods. showed high applicability. This statistical result proves that th...

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Abstract

The invention provides multiple PCR primers for detecting polymyxin drug resistance genes. The multiplex PCR primers are composed of the following five primer pairs, including the primer pair shown inSEQ ID No:1 and SEQ ID No:2, the primer pair shown in SEQ ID No:3 and SEQ ID No:4, the primer pair shown in SEQ ID No:5 and SEQ ID No:6, the primer pair shown in SEQ ID No:7 and SEQ ID No:8 and the primer pair shown in SEQ ID No:9 and SEQ ID No:10. The invention further provides a detection kit and a detection method for detecting the polymyxin drug resistance genes. The primers are designed based on a gene sequence of mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5, and the drug resistance genes corresponding to the sizes of different PCR products are distinguished. After a reaction system and conditions are optimized, finally, a complete multi-PCR reaction system is determined. The quick and effective detection method is provided for simultaneously detecting the polymyxin drug resistance genes-a mcr gene family and has high sensitivity and specificity.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a multiplex PCR primer, a kit and a detection method for detecting polymyxin drug-resistant genes. Background technique [0002] Polymyxin is an antibiotic that has been used for a long time in clinical treatment. It was first isolated and extracted in 1947, and then entered clinical use. However, due to the existence of neurotoxicity and nephrotoxicity of polymyxin, its clinical use has gradually decreased. In recent years, with the gradual increase of multidrug-resistant Gram-negative bacteria, polymyxins are considered to be the "last line of defense" against multidrug-resistant Gram-negative bacteria. [0003] In 2016, Chinese scientists were the first to report the colistin resistance gene mcr-1, which can be transferred horizontally between bacteria through a plasmid. The last line of defense” poses a huge threat. mcr-1 is a gene encoding phospho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 冯友军
Owner 青岛智烨生物科技有限公司
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