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PR-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit and detection method of swine transmissible gastroenteritis virus

A detection kit and infectious technology, applied in the field of biological detection, can solve the problems of difficult cell separation and culture, high cost of virus detection, difficulty in virus diagnosis, etc., and achieve the effect of good specificity, simple and practical method, and high sensitivity

Inactive Publication Date: 2014-04-23
BEIJING DAWEIJIA BIOTECH SHARE CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The detection methods of porcine transmissible gastroenteritis virus include a series of antigen and antibody detection techniques such as virus isolation and identification, immunofluorescence antibody, electron microscope observation, RT-PCR, ELISA and colloidal gold technology, while the cell separation of porcine transmissible gastroenteritis virus It is very difficult to cultivate, and the use of immunofluorescence antibody technology, electron microscope technology, etc. to detect viruses is costly and requires special instruments. These technologies cannot meet the needs of rapid clinical detection of porcine transmissible gastroenteritis virus antigens
Since porcine transmissible gastroenteritis virus recessive infection in pig farms is relatively common, and it is difficult to diagnose the virus in clinic, therefore, it is necessary to develop a method with high sensitivity, good specificity, simple and practical method, which can directly and quickly detect the virus. The kit for detecting porcine transmissible gastroenteritis virus pathogens from pig feces and intestinal samples will have great application value

Method used

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  • PR-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit and detection method of swine transmissible gastroenteritis virus
  • PR-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit and detection method of swine transmissible gastroenteritis virus
  • PR-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit and detection method of swine transmissible gastroenteritis virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation and use method of embodiment 1 kit

[0022] 1. Design and synthesis of primers

[0023] According to the gene sequence of transmissible gastroenteritis virus in GenBank as a template, a pair of specific primers were designed with the software Primer5.0, the upstream primer P1: 5'-AGACAAACTCGCTATCGCATGG-3'; the downstream primer P2: 5'-TCTTGTCACATCACCTTTACCTGC-3 ', a 531bp DNA fragment can be amplified after the PCR reaction.

[0024] 2. Sample preparation

[0025] Take 0.5g of intestinal tract and feces samples from porcine transmissible gastroenteritis virus-infected pigs, cut them into pieces and place them in a grinder, add 1mL of PBS and grind them, put the grinding solution in a 1.5ml sterilized centrifuge tube, and centrifuge at 3000r / min After 15 minutes, take 300 μl of the supernatant and put it in a 1.5ml sterilized centrifuge tube as the sample to be tested; take 300 μl each of the positive control, negative control, and sample to be tested, ...

Embodiment 2

[0032] The specificity and sensitivity test of embodiment 2 kit

[0033] 1, specificity test: use test kit of the present invention to porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) swine fever virus (CSFV), porcine parvovirus respectively Virus (PPV) porcine pseudorabies virus (PRV) and porcine transmissible gastroenteritis virus (TGEV) positive disease materials were tested, the results (see figure 2 ) shows that this kit can specifically detect porcine transmissible gastroenteritis virus, while the detection results of positive disease materials of other viruses are all negative. It can thus be shown that the detection kit of the present invention has good specificity and can accurately determine whether porcine transmissible gastroenteritis virus exists in the sample.

[0034] 2. Sensitivity test: After measuring the concentration of the prepared RNA sample with an ultraviolet analyzer, add 5 μ...

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Abstract

The invention discloses a PR-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit of swine transmissible gastroenteritis virus, which comprises an RT reaction solution, a positive control, a negative control, DEPC (diethylpyrocarbonate) water, 2*Taq PCR Master Mix and a primer mixed solution. Compared with an existing detection technology, the PR-PCR detection kit of the swine transmissible gastroenteritis virus has the advantages of high sensitivity, good specificity, simple and practical and the like, and can be used for rapidly detecting transmissible gastroenteritis of swine virus in various clinical samples on a large scale.

Description

technical field [0001] The invention relates to a PR-PCR detection kit for porcine transmissible gastroenteritis virus and a detection method thereof, belonging to the technical field of biological detection. Background technique [0002] Porcine transmissible gastroenteritis of swine virus (TGEV) belongs to the Coronaviridae family of Coronaviridae. Like porcine epidemic diarrhea virus and porcine rotavirus, it is one of the important pathogens that cause viral diarrhea in piglets. The occurrence of transmissible gastroenteritis (TGE) disease. TGE is an acute, highly contagious disease characterized by vomiting, severe diarrhea, dehydration, and high mortality in newborn piglets up to 2 weeks of age. TGE has occurred widely all over the world. Pigs of different ages and breeds are susceptible to the disease. The mortality rate of 2-week-old piglets exceeds 90%, which has caused huge economic losses to the pig industry. [0003] The detection methods of porcine transmissib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/70C12Q1/686C12Q2531/113
Inventor 董剑辉张志刚张文利张晓杰金忠辉
Owner BEIJING DAWEIJIA BIOTECH SHARE CO LTD
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