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Norovirus traceability method and its primers, kit and application

A kit and virus technology, applied in the field of microbial detection, to achieve the effect of accurate measurement results and high sensitivity

Inactive Publication Date: 2020-05-08
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is no report on the application of PCR-DGGE technology to the traceability of norovirus

Method used

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  • Norovirus traceability method and its primers, kit and application
  • Norovirus traceability method and its primers, kit and application
  • Norovirus traceability method and its primers, kit and application

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specific Embodiment approach

[0048] This embodiment provides a primer for norovirus traceability, which includes:

[0049] Upstream primer F: 5'-ATGTTCMGMTGGATGMGATTC-3';

[0050] Downstream primer R: 5'-TMGACGCCATCWTCATTYAC-3';

[0051] Downstream primer GC-R: 5'-CGCCCGGGGCGCGCCCCGGGGCGGGGCGGGGGCGCGGGGG

[0052] GTMGACGCCATCWTCATTYAC-3'.

[0053] This embodiment also provides a test kit for norovirus traceability method, said test kit includes the above-mentioned primers, and the formula of each said test kit is:

[0054] Upstream primer F, 20μM, 0.6μL; Downstream primer R, 20μM, 0.3μL; Downstream primer GC-R, 20μM, 0.3μL; dNTP, 2.5mM, 1.6μL; rTaq, 5U / μL, 0.8μL; 10×PCR buffer , 8 μL; ddH 2 O, 80 μL.

[0055] This embodiment also provides a PCR-DGGE-based norovirus traceability method utilizing the above kit, comprising the steps of:

[0056] (1) enriching Norovirus and extracting the DNA of said Norovirus, wherein the method for enriching Norovirus comprises the following steps:

[0057] Take 25g ...

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Abstract

The invention provides a method, a primer and a kit for tracing the source of Norovirus, and applications of the kit. The method comprises: (1) enriching norovirus, extracting the genomic RNA of the norovirus, and carrying out reverse transcribing to obtain cDNA so as to be spare; (2) designing the following primer by using the genomic cDNA obtained in the step (1) as a template, and carrying out PCR amplification on the genomic DNA; (3) carrying out denaturing gradient gel electrophoresis treatment on the PCR amplification product obtained in the step (2), analyzing the obtained gel map, recovering the gel strip, designing the following primer by using the recovering product of the gel strip as a template, and carrying out PCR amplification; and (4) carrying out gel cutting recovery on the PCR amplification product obtained in the step (3), purifying, carrying out cloning sequencing on the PCR product, logging in the Genbank, and carrying out sequencing comparison to obtain the genotype of the norovirus. According to the present invention, the method has advantages of accurate, stable and reliable determination result, high sensitivity, and low detection cost.

Description

technical field [0001] The invention belongs to the field of microbial detection, in particular to a method for tracing the origin of norovirus based on PCR-DGGE and its primers, kit and application. Background technique [0002] The quality and safety of food has become an important indicator related to the vital interests of the people, social stability and the expansion of food export earnings, and food-borne diseases caused by food-borne viruses are one of the main factors affecting food safety. Food quality problems and productivity shrinkage caused by foodborne viruses not only cause economic losses, but also restrict the development of my country's food export trade. The scare caused by mad cow disease in the UK in 1996 promoted the direction of research on the traceability system. The Danish pork salmonella contamination incident and the Scottish E. coli incident caused EU consumers to lack information on government food safety supervision, which all promoted the est...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2565/125
Inventor 杨向莹张捷张文玲李小林王琳畅晓晖万晓楠陈广全
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT