Primer and kit for simultaneously detecting PEDV, PRRSV, TGEV and PoRV and use method thereof

A technology of TGEVP-R and TGEVP-F, applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low accuracy, poor specificity, and time-consuming, and achieve high sensitivity and specificity. The effect of strong performance and broad application prospects

Pending Publication Date: 2020-09-11
河北三狮生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the problems of long time-consuming, poor specificity and low accuracy in existing methods for detecting porcine transmissible gastroenteritis virus, porcine blue e

Method used

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  • Primer and kit for simultaneously detecting PEDV, PRRSV, TGEV and PoRV and use method thereof
  • Primer and kit for simultaneously detecting PEDV, PRRSV, TGEV and PoRV and use method thereof
  • Primer and kit for simultaneously detecting PEDV, PRRSV, TGEV and PoRV and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Primer design:

[0031] According to the MK841495 gene of PEDV, the AY150312 gene of PRRSV, the AF209745 gene of TGEV, and the MF278302 gene of PORV registered in Genbank, primers were designed respectively.

[0032] The primers used for PCR reaction screened in table 1

[0033]

Embodiment 2

[0035] Multiplex PCR detection method:

[0036] (1) Genome extraction: extract the virus genome according to the operating instructions of the Double Helix Animal Genome DNA / RNA Extraction Kit, and store it at -20°C for later use;

[0037] (2) Reverse transcription (synthesis of cDNA)

[0038] Prepare the reverse transcription reaction system, add 16 μL of RNA template and 4 μL of 5×RNA reverse transcription reaction mixture (including Buffer, dNTP, M-MLV, RNase inhibitor, universal reverse transcription primer, etc.) , after the reaction, reverse transcriptase was inactivated at 85°C to obtain cDNA for later use;

[0039] (3) High-throughput PCR amplification

[0040] Add 3.3 μL of sterilized water, 12.5 μL of 2×PCR amplification MIX, 0.2 μL of upstream and downstream primers for porcine transmissible gastroenteritis virus (10 μmol / L) and 0.2 μL of upstream and downstream primers for porcine PRRS virus in the PCR reaction tube. (10 μmol / L) each 1.0 μL, porcine epidemic dia...

Embodiment 3

[0044] 3.1 Specificity test

[0045]According to the method for embodiment 2, detect porcine transmissible gastroenteritis virus gene (TGEV), porcine blue ear disease virus (PRRSV), porcine epidemic diarrhea virus gene (PEDV), porcine rotavirus gene (PoRV), swine fever porcine circovirus type 2 (PCV2), porcine Japanese encephalitis virus (JEV), porcine bocavirus (PBoV), porcine deltacoronavirus (PDCoV) and porcine pseudorabies virus (PRV)10 virus, and sterilized water as a negative control, to verify the specificity of the primers of the present invention.

[0046] Use a DNA / RNA extraction kit to extract the genomic RNA of TGEV, PRRSV, PEDV, PoRV, CSFV, JEV, and PDCoV, and perform reverse transcription of the extracted genomic RNA to obtain cDNA, and use a DNA / RNA extraction kit to extract PCV2 , pBoV and PRV genomic DNA, and use the cDNA obtained by reverse transcription and the extracted DNA as templates to perform PCR amplification detection, and detect according to the me...

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Abstract

The invention relates to the technical field of virus detection, and particularly discloses a primer and kit for simultaneously detecting PEDV, PRRSV, TGEV and PoRV and a use method thereof. The PCR detection primer provided by the invention can be used for simultaneously detecting four kinds of viruses including the PEDV, the PRRSV, the TGEV and the PoRV, has no cross amplification reaction withswine fever virus, swine Japanese encephalitis virus, swine Bocavirus, porcine circovirus 2, swine delta circovirus, porcine pseudorabies virus and other swine viruses, and has relatively strong specificity; and the primer is high in sensitivity, the lowest detection concentrations of the primer to the PEDV, the PRRSV, the TGEV and the PoRV are 9.12x10<5> pg/[mu]L, 2.48x10<3> pg/[mu]L, 7.55x10<5>pg/[mu]L and 8.23x10<5> pg/[mu]L respectively, and the detection accuracy is 100%.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a primer, a kit and a use method for simultaneously detecting PEDV, PRRSV, TGEV and PoRV. Background technique [0002] Porcine transmissible gastroenteritis is a highly contagious gastrointestinal infectious disease caused by porcine transmissible gastroenteritis virus (TGEV), characterized by vomiting, watery diarrhea and dehydration, and is listed as a category B animal disease by the OIE . Pigs of all ages are susceptible, especially piglets within 10 days of age have the highest morbidity and mortality, and are mostly endemic, and outbreaks can occur in new areas. [0003] Porcine reproductive and respiratory syndrome is an acute infectious disease caused by porcine blue ear virus, also known as porcine reproductive and respiratory syndrome virus (PRRSV). The main clinical features are abortion, stillbirth, mummified fetus, weak fetus, respiratory Difficulties, etc...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 李清扬李一鸣焦义然张婵石磊时国强范葶莉李翀王琨
Owner 河北三狮生物科技有限公司
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