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Specific primer combination and probe combination for detecting four glycopeptide drug resistance genes in enterococcus bacteria and application thereof

A drug-resistant gene, Enterococcus technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc. Achieving significant promotion value and high specificity

Inactive Publication Date: 2019-07-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PCR method also has shortcomings such as the uncertainty of judging the results only based on the size of the amplified product fragments, and the complexity of sequencing data analysis, which affects the reliability of the detection results and its wide promotion.

Method used

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  • Specific primer combination and probe combination for detecting four glycopeptide drug resistance genes in enterococcus bacteria and application thereof
  • Specific primer combination and probe combination for detecting four glycopeptide drug resistance genes in enterococcus bacteria and application thereof
  • Specific primer combination and probe combination for detecting four glycopeptide drug resistance genes in enterococcus bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Coverage and specificity of primer combinations and probe combinations for four glycopeptide drug resistance genes

[0045] This example illustrates the coverage and specificity of the primer combination and probe combination for detecting four glycopeptide drug resistance genes described in the present invention through the following steps.

[0046] Step 1: Download the reference sequences of the four glycopeptide drug resistance genes vanA, vanB, vanD and vanM involved in the present invention from the authoritative database of antibiotic resistance—The Comprehensive Antibiotic Resistance Database (CARD): M97297( vanA), KF823969(vanB), AY082011(vanD) and FJ349556(vanM).

[0047] Step 2: Submit the reference sequences of the above four drug-resistant genes to NCBI for online Blast comparison, and the comparison database is a non-redundant nucleic acid database (version 20190315). Among them, the vanA gene reference sequence M97297 obtained a total of 253 al...

Embodiment 2

[0060] Embodiment 2, the detection specificity of the specific primer probe combination of the present invention

[0061] The primers and probes used in this example were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0062] 1. Preparation of samples to be tested

[0063] In this example, plasmid DNAs containing four glycopeptide drug resistance genes vanA, vanB, vanD and vanM were used as test samples to test the detection specificity of the primer-probe combination described in the present invention.

[0064] The preparation method of each of the above-mentioned plasmids is as follows:

[0065]Sample 1 to be tested is a plasmid containing the drug-resistant gene vanA: replace the sequence between the HindIII and EcoRI sites of the pUC18 plasmid with the sequence shown in the vanA gene reference sequence M97297 to obtain a recombinant plasmid that contains the drug-resistant gene vanA plasmid;

[0066] Sample 2 to be tested contains a plasmid containing the drug...

Embodiment 3

[0127] Embodiment 3, detect enterococcus bacterial strain with specific primer probe combination of the present invention

[0128] In this example, the collected glycopeptide drug-resistant strains, specifically vancomycin-resistant Enterococcus faecalis (Enterococcus faecalis), faecium (Enterococcus faecium), and glycopeptide drug-sensitive strains were tested for vanA, The detection of vanB, vanD and vanM drug resistance genes is to illustrate that the specific primer probe combination provided by the present invention can be used to detect the detection of the above four drug resistance genes in glycopeptide drug resistant strains.

[0129] details as follows:

[0130] 1. Samples to be tested

[0131] In this example, the specific primer-probe combination described in the present invention was used to detect vancomycin-resistant Enterococcus faecalis and Enterococcus faecium strains, and vancomycin-sensitive strains respectively.

[0132] It was confirmed by sequencing th...

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Abstract

The invention discloses a specific primer combination and a probe combination for detecting four glycopeptide drug resistance genes vanA, vanB, vanD and vanM in enterococcus bacteria, comprising a primer combination for specific amplification of four glycopeptide drug resistance genes and further comprising specific probes for detecting the four glycopeptide drug resistance genes. A method for detecting the four glycopeptide drug resistance genes by using the specific primer and probe combinations is further provided. The combinations have high specificity and coverage, can be used not only for detecting the drug resistance genes vanA, vanB, vanD and vanM in human infected samples but also for suggesting resistance degree to vancomycin and teicoplanin, and provides a reference basis for clinical anti-infective therapy medication. At the same time, the development of glycopeptides drug resistance is monitored. The combinations can also be used for monitoring the distribution and transfer of the four glycopeptide drug resistance genes in animal and environmental samples.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a combination of specific primers and probes for detecting four glycopeptide drug resistance genes in Enterococcus bacteria and its application. The involved VanA, VanB, VanD and VanM operons were all isolated from bacteria of the genus Enterococcus. The affinity of glycopeptides to peptidoglycan precursors ultimately manifests as high resistance to vancomycin and to teicoplanin (except VanB). Background technique [0002] Glycopeptide antibiotics are a class of antibiotics with a heptapeptide structure, composed of amino acids and sugar residues, chlorine atoms, methyl groups and / or lipid chains, through the d-ala-d- Ala end to inhibit the synthesis of peptidoglycan, so that bacteria can not synthesize the cell wall normally, so as to achieve antibacterial effect. Important glycopeptide antibiotics used to treat human infections include the first-generation drugs Vancomycin (appro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816C12Q1/689C12Q1/04C12N15/11
CPCC12Q1/6816C12Q1/689C12Q2531/113
Inventor 张琼孟晓华刘芳周佳维吴灵娇倪淑君胡绍华苏琨楷姚坚刁宏燕
Owner ZHEJIANG UNIV
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