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Fourfold RT-PCR detection primer and reagent kit of four porcine epidemic diarrheaviruses

A technology of RT-PCR and detection primers, which is applied in the field of quadruple RT-PCR detection primers and kits, can solve the problems of mixed infection and inapplicable virus detection, and achieve strong specificity

Active Publication Date: 2019-08-06
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Clinically, the above four porcine diarrhea virus infections are common, and mixed infections are common. Although the traditional single RT-PCR technology is fast and specific, it is not suitable for large-scale clinical virus detection.

Method used

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  • Fourfold RT-PCR detection primer and reagent kit of four porcine epidemic diarrheaviruses
  • Fourfold RT-PCR detection primer and reagent kit of four porcine epidemic diarrheaviruses
  • Fourfold RT-PCR detection primer and reagent kit of four porcine epidemic diarrheaviruses

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Experimental program
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Effect test

Embodiment 1

[0053] Experimental materials: positive samples of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine group A rotavirus (PoRV) and porcine delta coronavirus (PDCoV) were from Anhui Agricultural University Zoonotic Diseases Laboratory, RNA extraction kit (column passing method) was purchased from Beijing Yisenbao Biotechnology Co., Ltd., 2×Taq PCR MasterMix was purchased from TIANGEN Company, Trans1-T1 Phage Resistant chemically competent cells and reverse transcription kit and pEASY - The T1Clonging Kit vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd., the agarose gel DNA recovery kit was purchased from TIANGEN Company, and the plasmid mini-prep kit was purchased from TIANGEN Company, etc.

[0054] experimental method:

[0055] 1. Collection and processing of disease materials

[0056] Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine group A rotavirus (PoRV)...

Embodiment 2

[0079] Sensitivity experiment of a single RT-PCR reaction

[0080] Measure the positive plasmid concentration of four kinds of viruses, use five distilled water to carry out 10-fold comparative dilution of four virus positive plasmids respectively, from 10 -1 diluted to 10 -9 A total of 9 dilutions. For the four viruses, 9 dilutions of the template of a single virus were used for the reaction. Four kinds of viruses TGEV, PEDV, PORV and PDCoV were tested for sensitivity respectively, and a negative control group was set at the same time.

[0081] Sensitivity test of RT-PCR of PEDV: use the optimized PEDV RT-PCR reaction to carry out the sensitivity test, the PEDV positive plasmid that has measured the good concentration is done tenfold ratio dilution, and the dilution factor is 10 -1 -10 -10 . The results of PEDV single PCR sensitivity experiment are as follows: Figure 8 (M: DL 2000 Marker; +: original concentration of PEDV plasmid; 1-10: ten-fold serial dilution 10 -1 ...

Embodiment 3

[0086] Establishment of Multiplex RT-PCR for Four Porcine Diarrhea Viruses

[0087] (1) Optimization of the annealing temperature of the quadruple RT-PCR method for four porcine diarrhea viruses

[0088] Taking TGEV, PEDV, PORV and PDCoV four virus positive plasmids as templates, choosing different temperatures, and testing the four viruses at the same time, it proves that the protection temperature of this application has the best effect, and the amplification effect of other temperatures is poor or ineffective. Amplify. In this experiment, 50°C, 52°C, 54°C, 56°C, and 58°C were selected as the test annealing temperature, and the optimization results of the annealing temperature for the quadruple RT-PCR detection system are as follows: Figure 12 (M: DL 2000 Marker; -: negative control; 1-5: 50°C, 52°C, 54°C, 56°C, 58°C), when the annealing temperature is 56°C, the quadruple RT-PCR detection method The amplification effects on the four viruses are the best, and the bands are...

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Abstract

The invention relates to a detection primer and reagent kit of four RT-PCR of porcine epidemic diarrheaviruses, transmissible gastroenteritis of swine, porcine rotaviruses and porcine deltacoronaviruses, and belongs to the technical field of molecular detection. The fourfold RT-PCR detection primer comprises detection primers PEDV F and PEDV R of porcine epidemic diarrhea viruses, detection primers TGEV F and TGEV R of the transmissible gastroenteritis of swine, detection primers PoRV F and PoRV R of the porcine rotaviruses and detection primers PDCoV-F and PDCoV-R of the porcine delta coronaviruses. The primer is high in specificity, has repeatability, is high in sensitivity and is high in clinical reliability.

Description

technical field [0001] The invention relates to the technical field of molecular detection, in particular to quadruple RT-PCR detection primers and kits for porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine group A rotavirus and porcine deltacoronavirus. Background technique [0002] Porcine epidemic diarrhea disease is an acute, highly contagious disease caused by porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV). The rate can reach 100%. The continuous outbreak of porcine epidemic diarrhea disease has brought huge economic losses to our country. Transmissible gastroenteritis of swine (TGE) is a highly contagious disease caused by porcine transmissible gastroenteritis virus (TGEV), which is susceptible to pigs of all ages and affects piglets most severely , with a 100% fatality rate. The disease occurs in every pig-raising country in the world, and has brought great economic losses to the world's pig-raising indus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/156C12Q2537/143
Inventor 孙裴李亮周亭宇秦娟李杰占松鹤王军高亚成
Owner ANHUI AGRICULTURAL UNIVERSITY
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