Study On The Differential Detection Method For Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED),a highly contagious disease,which was characterized by vomiting,acute diarrhea and severe dehydration of newborn piglets,was caused by porcine epidemic diarrhea virus(PEDV)and has caused huge economic losses to the pig industry.Clinically,the symptoms of the diseases caused by PEDV,porcine transmissible gastroenteritis virus(TGEV)and porcine rotavirus(PRoV)are very similar,and it is difficult to distinguish them.So it is necessary to diagnose them by laboratory test.It is very important to establish a rapid and specific detection method of PEDV,TGEV and PRoV for prevention and control of swine diarrhea diseases.1.Establishment and application of a multiplex RT-PCR assay for detection of PEDV,TGEV and PRoVIn this study,a multiplex RT-PCR assay was established to differentially detect PEDV,TGEV and PRoV after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus(CSFV),porcine foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV)and porcine circovirus type 2(PCV2).The detection limits of PEDV,TGEV andPRoV standard recombinant plasmids were 1.41×103?1.41×102 and 1.41×103 copies/?L,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42(22.11%)samples were positive for PEDV,58(30.53%)samples for TGEV and 34(17.89%)sample for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.2.Establishment and application of a multiplex RT-PCR assay fordifferential detection of wild-type and vaccine strains of PEDVA multiplex RT-PCR assay was established for differential detection of classical,variant and vaccine strains of PEDV after optimization of the reaction conditions.Three pairs of primers were designed for specifically amplifying wild-type(classical and variant)and vaccine strains PEDV,respectively,of which the first pair of primers was used for amplification of 198 bp and 148 bp ORF3 gene fragments of wild-type(classical and variant)and vaccine strains,the second used for amplification of 429 bp S gene fragment of classical and vaccine strains,and the third used for amplification of 274 bp S gene fragments of variant strain.The assay could specifically amplify PEDV,but not other swine pathogens such as FMDV,CSFV,PRRSV,TGEV,PRoV,PCV,PRV and so on.Thedetection limit of PEDV standard recombinant plasmids was 2.62×102 copies/?L.The repeated test acquired uniform results.The assay was used to detect a total of 336 clinical samples and 120 were positive for PEDV,of which 96 were positive for variant strain,21 for vaccine strains,and 3 for variant and vaccine strains.The results indicated that the established multiplex RT-PCR assay was sensitive,specific and repeatable,and could be used as a good tool for differential detection and epidemiological investigation of PEDV.In conclusion,this study successfully established two rapid detection methods of PEDV,which provided an effective technical platform for differential diagnosis of diarrhea virus.