Establishment And Application Of Six RT-PCR Assay For Detection Of Porcine Viral Diarrhea And Isolation And Identification Of Porcine Epidemic Diarrhea Virus

Viral diarrhea severely damages the pig industry,causing tremendous economic loss worldwide.The most common viruses causing diarrhea are porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine rotavirus A(PRV-A).In the past years,some newly emerged viruses like porcine kobuvirus(PKV),porcine sapovirus(PSaV),and porcine deltacoronavirus(PDCoV)were also discovered.It is critically important but hard to make a differential diagnosis of porcine viral diarrhea due to diverse pathogens in the gastrointestinal tract and high similarity in clinical signs and pathological changes caused by enteric viral infection.To develop a multiplex RT-PCR for rapid detection and differential diagnosis of viral diarrhea caused by the above viruses,specific primer sets targeting the N genes of TGEV,PEDV and PDCoV,the VP7 gene of PRV-A,and the polyproteins of PKV and PSaV were designed based on highly conserved regions.The multiplex RT-PCR was successfully developed and optimized for the detection of 6 diarrheal viruses in one reaction vial spontaneously,showed high sensitivity and specificity.Lastly,the multiplex RT-PCR assay was employed to analyze 382 field diarrheal samples.The results indicated that PDCoV,PSaV and PEDV were do minantly circulating in pig farms of China between October 2015 and April 2017,with co-infections by dual or triple swine enteric viruses.The multiplex RT-PCR specifically target main swine enteric viruses circulating in Chinese pig farms and will provide a valuable tool for the clinical diagnostic laboratory.Previous results showed that PEDV was prevalent in pig farms in China.Therefore,multiple RT-PCR results showed that PEDV positive genes were amplified by primers targeting PEDV N gene,and the target fragment was successfully amplified.a new PEDV strain was found from the diarrhea specimens collected from a pig farm located in Yantai,Shandong province.The whole geneome of the strain was sequenced.The S gene and spike protein was also analyzed.All results demonstrated that this newly isolated PEDV strain had significant variations comparing to classic strains and was an epidemic PEDV strain.These results indicated that the vaccines developed from the classic PEDV strains may not work for the prevention and control of current epidemics of PED and new vaccines based on the epidemic PEDV strains are imperative.To prepare monoclonal antibodies against PEDV-N protein,this study adopted RT-PCR method from YT1401 pig epidemic diarrhea virus strains amplified its N protein gene fragments.Construction of prokaryotic expression recombinant plasmid pET30a-PEDV-N,positive plasmid into E.coli BL21 competent cells and to optimize conditions for inducing expression,SDS-PAGE,Western Blotting test.The results showed that SDS-PAGE detection results show that the expression product won about 57 kDa,and size conform to target protein,and has good immunological activity.