Establishment And Application Of Porcine Enterovirus Multiplex RT-PCR Assay And Complete Sequence Analysis For 17GXCZ-1 Strain Of Porcine Epidemic Diarrhea Virus

Diarrheal diseases caused by porcine enterovirus are common diseases in pigs,which can cause diarrhea,dehydration and weight loss in pigs of all ages.The clinical symptoms are similar and difficult to distinguish,resulting in high morbidity and high mortality of piglets,and causing serious economic losses to the pig industry.Among them,PEDV is one of the most serious diseases in pig industry,and the variant strain has been widely prevalent.It is necessary to have a deeper understanding of the molecular structure characteristics and genetic variation characteristics of PEDV.1.Establishment and application of multiplex RT-PCR detection method for porcine enterovirus.In this study,multiplex RT-PCR method was successfully established to detect PEDV,TGEV,PDCo V,PRo V and PKV simultaneously and rapidly.The minimum detection limit of samples was 7.56×10~1 copies/?L,which was highly specific,sensitive and repeatable.Total 171 samples from different regions of Guangxi province during 2017-2018 were collected and investigated the presence of PEDV,TGEV,PDCo V,PRo V and PKV by using the established multiplex RT-PCR method.The positive rates were 64.91%,19.30%,8.77%,12.87%and 63.74%,respectively.The results showed that the type of infection for porcine enterovirus in Guangxi region was mostly mixed infection(60.82%).Among them,PEDV and PKV have the highest positive rate of mixed infection(30.41%),followed by PEDV,TGEV and PKV(10.53%).2.Complete genome sequence analysis for 17GXCZ-1 strain of porcine epidemic diarrhea.In this study,the complete genome sequence of 17GXCZ-1 isolated from the laboratory was successfully obtained,with a total length of 28035bp,and the sizes of 5'UTR,ORF1,S,ORF3,E,M,N,and 3'UTR genes were 292,20345,4158,675,231,681,1326,and 334bp,respectively.The homology between17GXCZ-1 and the reference strains in the nucleotide sequence of complete sequence was 96.3?98.2%.The nucleotide homology between 17GXCZ-1 and the reference strains in the S gene was 93.4?98.1%,and the amino acid homology of the S gene was 92.8?98.7%.The nucleotide homology between17GXCZ-1 and the reference strains in the ORF3 gene 95.3?99.6%,and the amino acid homology of the ORF3 gene was 89.0?100.0%.According to the results of genetic evolution analysis,17GXCZ-1 is G2-a type variant strain,and is closely related to AJ1102 strain.The prediction results for the transmembrane domain composition,the maximum and minimum hydrophobicity,the signal peptide cleavage site and the potential N-glycosylation site of the S and ORF3proteins of 17GXCZ-1 strain were identical to those of the AJ1102 strain by four biological softwares.In summary,not only a scientific and effective detection method for the differential diagnosis of porcine enterovirus and the monitoring of mixed infection has been provided,but also an effective technical guarantee for clinical application has been provided in this study.And the genetic evolution of PEDV strains in Guangxi has been deeply analyzed,which laid a solid foundation for further research on infectious cloning of PEDV strains.