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Primer, probe, method and kit for detecting VRE (Vancomycin-resistant Enterococcus spp.) gene

A vancomycin intestine and primer probe technology, applied in the field of molecular biology, can solve the problems of difficulty in distinguishing different genotypes, low sensitivity, complicated operation, etc., and achieve the effects of high accuracy, strong specificity and high sensitivity

Inactive Publication Date: 2016-09-21
宁波基内生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The invention provides a primer, a probe, a method and a kit for detecting the gene of vancomycin-resistant enterococcus, which solves the problem that the detection of VRE in the prior art is not only cumbersome, time-consuming, labor-intensive, low in sensitivity, poor in repeatability, and difficult to distinguish by traditional methods. different genotypes, and the technical problems of not being able to understand the molecular biological mechanisms of resistance

Method used

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  • Primer, probe, method and kit for detecting VRE (Vancomycin-resistant Enterococcus spp.) gene
  • Primer, probe, method and kit for detecting VRE (Vancomycin-resistant Enterococcus spp.) gene
  • Primer, probe, method and kit for detecting VRE (Vancomycin-resistant Enterococcus spp.) gene

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Embodiment 1

[0024] The embodiment of the present invention provides a primer and probe for detecting the gene of vancomycin-resistant enterococcus, the nucleotide sequence of the primer for detecting the 16S rDNA of the enterococcus gene is shown in SEQ ID NO: 2-3, and the nucleotide sequence for detecting the 16S rDNA of the enterococcus gene The nucleotide sequence of the Taqman probe is shown in SEQ ID NO: 4; the nucleotide sequence of the primers for detecting the specific gene vanA of vancomycin-resistant enterococcus is shown in SEQ ID NO: 6-7, and the specificity of detecting vancomycin-resistant enterococcus The nucleotide sequence of the Taqman probe of the gene vanA is shown in SEQ ID NO: 8; the nucleotide sequence of the primers for detecting the specific gene vanB of vancomycin-resistant enterococci is shown in SEQ ID NO: 10-11, and the detection of vancomycin-resistant enterococcus The nucleotide sequence of the Taqman probe of the cocci-specific gene vanB is shown in SEQ ID N...

Embodiment 2

[0042] In the embodiment of the present invention, a method for detecting the gene of vancomycin-resistant enterococcus is provided, including:

[0043] Step 101, sample nucleic acid preparation to obtain a nucleic acid template;

[0044] Step 102, designing specific primers and fluorescently labeled probes for the enterococcus gene 16S rDNA, the vanA specific gene vanA of the vancomycin-resistant enterococcus, and the vanB specific gene vanB of the vancomycin-resistant enterococcus;

[0045] Wherein, the nucleotide sequence of primers for detecting enterococcus gene 16S rDNA is shown in SEQ ID NO: 2-3, the nucleotide sequence of Taqman probe for detecting enterococcus gene 16S rDNA is shown in SEQ ID NO: 4; the detection of vancomycin resistance The nucleotide sequence of the primer for the specific gene vanA of enterococcus is shown in SEQ ID NO: 6-7, and the nucleotide sequence of the Taqman probe for detecting the specific gene vanA of vancomycin-resistant enterococci is s...

Embodiment 3

[0056] An embodiment of the present invention provides a kit for detecting the gene of vancomycin-resistant enterococci, including a nucleic acid extraction solution, a first primer-probe mixed solution, a second primer-probe mixed solution, a third primer-probe mixed solution, PCR reaction enzyme system, negative control substance, positive control substance and the packing box that separates and pack these reagent bottles or tubes collectively, wherein, the first primer-probe mixture is composed of deoxyribonucleoside triphosphate dN(U)TP, 16S rDNA gene upstream and downstream primers and a fluorescent label probe; the second primer probe mixture consists of deoxyribonucleoside triphosphate dN(U)TP, vanA gene upstream and downstream primers and a fluorescent label probe needle composition; the third primer-probe mixture is composed of deoxyribonucleoside triphosphate dN(U)TP, vanB gene upstream and downstream primers and a fluorescent labeling probe;

[0057] Wherein, the nu...

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Abstract

The invention relates to the technical field of molecular biology, and discloses a primer, a probe, a method and a kit for detecting a VRE (Vancomycin-resistant Enterococcus spp.) gene. Primers and fluorescent labeled probes are respectively designed aiming at a nucleic acid conservative region of 16S rDNA (ribosome Deoxyribose Nucleic Acid), vanA and vanB by adopting a Taqman probe real-time fluorescence PCR (Polymerase Chain Reaction) method; the 5' ends of 3 gene probes are all labeled by a fluorescent report group FAM, and 3' ends are all labeled by a fluorescent quenching group TAMRA. After the primer and the probe are prepared into a PCR detection mixed solution, enzyme and sample nucleic acid are added, an FAM channel on a fluorescent PCR instrument is selected for amplification, and detection on a target gene is realized through change of a fluorescent signal. The kit disclosed by the invention has the characteristics of high accuracy, strong specificity ad high sensitivity, and enterococcus and VRE in a urine sample can be rapidly and accurately detected.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer, a probe, a method and a kit for detecting the gene of vancomycin-resistant enterococci. Background technique [0002] Vancomycin-Resistant Enterococcus spp. (VRE) can cause urinary tract, abdominal cavity, pelvic cavity, surgical infection, endocarditis, meningitis and other diseases, with a fatality rate of 21.0% to 27.5%. The main nosocomial opportunistic pathogens in the range. Among them, Enterococcus faecalis and Enterococcus faecium are mainly pathogenic to humans, and the most common drug-resistant genotypes are vanA and vanB. As a common nosocomial infection, enterococci are multidrug-resistant bacteria that are naturally resistant to various antibacterial drugs such as cephalosporins, some fluoroquinolones, and aminoglycosides. Vancomycin-resistant enterococci can transfer their resistance genes to other pathogenic microorganisms. Therefore, it is ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2561/101
Inventor 王伟建倪剑锋包婷婷翁毅
Owner 宁波基内生物技术有限公司
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