Porcine Deltacoronavirus and swine transmissible gastroenteritis virus multiplex RT-PCR detection primer and detection method

A RT-PCR, coronavirus technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/test, etc., can solve the problems of complicated operation, difficult to effectively distinguish PDCoV infection, etc., to achieve the effect of rapid differential diagnosis

Active Publication Date: 2016-03-16
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, these methods are more complicated to operate, and it is diffic

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  • Porcine Deltacoronavirus and swine transmissible gastroenteritis virus multiplex RT-PCR detection primer and detection method
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  • Porcine Deltacoronavirus and swine transmissible gastroenteritis virus multiplex RT-PCR detection primer and detection method

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[0022] According to the N gene sequence of HKU15-155 strain of PDCoV and the N gene sequence of H155 strain of TGEV registered in GenBank, the present invention obtains its highly conserved specific sequence through sequence analysis, and designs two pairs of specific primers according to the specific conserved sequence . On this basis, a multiplex RT-PCR detection method for PDCoV and TGEV was established, and the method was evaluated and analyzed. The samples of disease materials submitted for inspection in Henan area were detected and analyzed, and the reliability of the method was further verified in practice, so as to provide a theoretical basis for veterinary clinical diagnosis and treatment.

[0023] Primers for multiplex RT-PCR detection of porcine deltacoronavirus and porcine transmissible gastroenteritis virus:

[0024] The primer sequences of porcine deltacoronavirus are as follows:

[0025] Upstream primer P1: 5'-ATGGCTACTGGCTGCGTTAC-3'

[0026] Downstream prime...

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Abstract

The invention discloses a porcine Deltacoronavirus (PDCoV) and swine transmissible gastroenteritis virus (TGEV) multiplex RT-PCR detection primer. The primer sequence of PDCoV is expressed as follows: upstream primer P1: 5'-ATGGCTACTGGCTGCGTTAC-3', downstream primer P2: 5'-GCGTTTCCTGGGCTGATT-3', and partial PDCoV gene segments are amplified by 383 bp. The primer sequence of TGEV is expressed as follows: upstream primer P3: 5'-CCCTCCAGCAAGGTTCAA-3', downstream primer P4: 5'-GCAACCCAGACAACTCCA-3', and partial TGEV gene segments are amplified by 229 bp. The detection limits of multiplex RT-PCR on PDCoV and TGEV are 4.05*101 copy per microliter and 5.47*102 copy per microliter respectively, and amplified results on porcine epidemic diarrhea virus, bocavirus, porcine reproductive and respiratory syndrome virus and porcine rotavirus are negative. Multiplex RT-PCR detection results of 57 clinical samples show that the positive rate of being infected with the two viruses at the same time is 1.75%, the PDCoV infection positive rate is 19.30%, and the TGEV infection positive rate is 1.75%.

Description

technical field [0001] The invention relates to a virus detection method, in particular to a multiple RT-PCR detection primer and detection method for porcine delta coronavirus and porcine transmissible gastroenteritis virus. Background technique [0002] Porcine Deltacoronavirus (Porcine Deltacoronavirus, PDCoV) belongs to Nidovirales (Nidovirales), Coronaviridae (Coronaviridae), Coronavirus (Coronavirus) Deltacoronavirus (Coronavirus), is a single-stranded positive-sense RNA virus with an envelope. As early as 2012, there were reports of PDCoV in the articles published by Woo et al.: They detected Delta coronavirus in mammalian and poultry disease materials collected in Guangdong and Hong Kong, among which it was detected in pig feces samples (169 samples) Nearly 10% of PDCoVs. In early 2014, a new porcine enteric coronavirus PDCoV was detected and reported for the first time in Ohio, USA. By the end of 2014, PDCoV infection cases had been found in 17 states in the United...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143Y02A50/30
Inventor 胡慧梁秀丽曹贝贝王亚宾魏战勇张君涛刘芳刘玲玲
Owner HENAN AGRICULTURAL UNIVERSITY
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