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Method for simultaneously detecting multiple RT-PCR of GETV, PEDV, TGEV, PDCoV and PoRV

A RT-PCR and multiplex technology, applied in the field of RT-PCR detection, can solve the problems of laborious and time-consuming, difficult to effectively distinguish virus types, etc., and achieve the effects of low homology, low cost, high amplification efficiency and sensitivity

Inactive Publication Date: 2018-12-07
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional detection methods of GETV, PEDV, TGEV, PDCoV and PoRV mainly include virus isolation and culture and serological methods, but it is laborious and time-consuming and it is difficult to effectively distinguish virus types

Method used

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  • Method for simultaneously detecting multiple RT-PCR of GETV, PEDV, TGEV, PDCoV and PoRV
  • Method for simultaneously detecting multiple RT-PCR of GETV, PEDV, TGEV, PDCoV and PoRV
  • Method for simultaneously detecting multiple RT-PCR of GETV, PEDV, TGEV, PDCoV and PoRV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Primer Design and Synthesis

[0058] (1) The design of the primer that is used to detect porcine getavirus:

[0059] The gene encoding Capsid protein in porcine getavirus (GETV) is crucial to the formation and survival of virus particles and is highly conserved, so the Capsid gene was selected as the target gene for detection of porcine getavirus. Using DNAMAN 6.0 to compare all Capsid reference gene sequences in the Genbank database, find relatively conserved fragments. Input the found conserved fragment of the Capsid gene into the Primer Premier 5.0 software, the length of the upper and lower primers is 19bp and 21bp, the Tm value is 60°C and 58.3°C, the G+C content is 57.9% and 47.6%, avoiding the formation of the hairpin structure of the primer itself, reducing the number of primers The formation of dimers and the reduction of non-specific amplification were used as parameters to design the upstream and downstream primer sequences for amplifying the Caps...

Embodiment 2

[0071] Embodiment 2: Nucleic acid extraction

[0072] According to the instruction manual of the TRIzon Reagent extraction reagent, the nucleic acid of the virus was extracted separately to obtain the RNA of each virus.

Embodiment 3

[0073] Embodiment 3: RT-PCR amplification

[0074] Using the extracted viral RNA as a template, it was reverse-transcribed into cDNA. The reverse transcription system is: 5×buffer 4μL, 10mmol / L dNTPs 1μL, 20μmol / L pd(N)6 (random hexamer primer) 0.5μL, 20μmol / L Oligo(dT) 0.5μL, 40U / μL RNA Enzyme inhibitor 0.5 μL, 200 U / μL reverse transcriptase (M-MLV) 0.5 μL and RNA template 13 μL; the reaction conditions of the reverse transcription are: 42° C. for 1 h, 95° C. for 10 min.

[0075] Using cDNA as template, carry out PCR amplification reaction. The reaction system of the PCR amplification reaction is: 2×Es TaqMasterMix 25 μL, mixed primer 2 μL, cDNA template 5 μL, ddH 2 0.18 μL, wherein the mixed primers are the primers for the detection of porcine getavirus, the primers for the detection of porcine epidemic diarrhea virus, the primers for the detection of porcine transmissible gastroenteritis virus, the detection of porcine del The primers for the tower coronavirus and the pr...

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Abstract

The invention discloses a multiple RT-PCR primer group for simultaneously detecting porcine gatahvirus (GETV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and porcine A rotaviruses (PoRV), which has a nucleotide sequence as shown in SEQ ID NO:1-SEQ ID NO:10. The invention further discloses a multiple RT-PCR detection method for detecting GETV, PEDV, TGEV, PDCoV and PoRV from a sample in one time by utilizing the multiple RT-PCR primer group. Compared with an existing conventional RT-PCR, the detection method has strong specificity and high sensitivity, can realizesimultaneous identification of five viruses including GETV, PEDV, TGEV, PDCoV and PoRV, and has accurate detection result and high detection efficiency.

Description

technical field [0001] The invention belongs to the technical field of RT-PCR detection, in particular to a multiple RT-PCR method for simultaneously detecting GETV, PEDV, TGEV, PDCoV and PoRV. Background technique [0002] Porcine Getavirus (Getah virus, GETV) belongs to the Togaviridae Alphavirus genus, and is a zoonotic arbovirus that can pass through mosquito vectors, aerosols, and infected animal tissues / organs, secretions, and It can be transmitted horizontally through excrement and other media, and can also be transmitted vertically through the placenta of animals, causing human and animal infection and disease in some animals. Antibody neutralization tests have confirmed that the positive rate of GETV antibodies in human serum samples can reach more than 20%, and accordingly it is listed as a zoonotic disease, although so far there is no report of the virus infecting humans and causing clinical disease. [0003] Porcine epidemic diarrhea virus (Porcine Epidemic Diar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2537/143C12Q2521/107
Inventor 王川庆常洪涛王傲杰陈陆周峰李永涛王新港杨霞崔丹丹王新卫杜季梅刘红英赵军
Owner HENAN AGRICULTURAL UNIVERSITY
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