Study On The Rapid Differential Methods For Detection Of Four Porcine Diahrrea Viruses And Molecular Epidemiology Of Porcine Epidemic Diahrrea Virus

1.Establishment and application of a multiplex RT-PCR for simultaneous detection of PEDV,TGEV,PDCoV and PRoVTo establish a rapid method for simultaneous detection of PEDV,TGEV,PDCoV and PRoV,four pairs of specific primers were designed according to gene sequences for amplifying PEDV N gene,TGEV M gene,PDCoV N gene and PRoV VP6 gene,respectively.After optimizing the reaction conditions,a multiplex RT-PCR for simultaneous detection of PEDV,TGEV,PDCoV and PRoV was established.The results showed that the assay could specifically amplify the target fragments from PEDV,TGEV,PDCoV and PRoV,but could not amplify the target fragments from others virus.The lowest detection limits of PEDV,PDCoV,PRoV and TGEV were 1.57×10~2,1.57×10~2,1.57×10~2,1.57×10~3 copies/?L,respectively.The repeatability test under the same reaction conditions resulted in uniform and consistent results.The established assay was used to detect 270 clinical diarrhea samples collected from Guangxi province,and the positive rates of PEDV,PDCoV,TGEV,PRoV were 15.56%,10.74%,11.48%,1.85%,respectively.There existed co-infections among four viruses.The results indicated that the multiplex RT-PCR for detection of PEDV,PDCoV,TGEV and PRoV were successfully established and could be used for differential detection and epidemiological investigation of these pathogens.2.Establishment and application of a multiplex TaqMan real-time RT-PCR for differential detection of PEDV,TGEV,PDCoV and PRoVTo develop an assay for differential detection of PEDV,TGEV,PDCoV and PRoV,four pairs of specific primers and TaqMan probes were designed for PEDV N gene,TGEV M gene,PDCoV M gene and PRoV VP6 gene,respectively.After optimizing the reaction conditions,a multiplex TaqMan real-time RT-PCR method for simultaneous detection of PEDV,TGEV,PDCoV and PRoV was established.The results showed that the established method could only specifically detected PEDV,TGEV,PDCoV and PRoV,and could not detect other major porcine viruses.The assay was highly sensitive with detection limit of 2.06×10~2 copies/?L for PEDV,2.06×10~2 copies/?L for TGEV,2.06×10~1 copies/?L for PDCoV and 2.06×10~3 copies/?L for PRoV.The assay had good repeatability with variation coefficients of intra-and inter-assays less than 1.1%.The multiplex TaqMan real-time RT-PCR was used to detect 243 diarrhea samples and the positive rates of PEDV,TGEV,PDCoV and PRoV were 11.93%,15.64%,9.05%,9.47%,respectively,and there existed mixed infection.The results indicated that the established method could provide a rapid,specific,sensitive and efficient technique for differential detection and epidemiological investigation of PEDV,TGEV,PDCoV and PRoV.3.Molecular epidemiology of PEDV during 2017-2019 in Guangxi Province,ChinaTo understand the genetic variation of the epidemic strains of PEDV in Guangxi province,clinical diahrrea samples collected from Guangxi were detected by the above-mentioned established multiplex RT-PCR and S,M,N genes were amplified,sequenced and analyzed from partial PEDV positive samples.The results showed that of 1454 samples detected during 2017-2019,157 samples(10.80%)were positive.After amplifying,cloning and sequencing,23 S gene sequences,25 M gene sequences and 25 N gene sequences of PEDV were acquired.The homology analysis revealed that the nucleotide and amino acid homology of S,M,N genes were 90.7%-100%and 85.0%-100%?97.5%-100%and 97.4%-100%?96.3%-100%and 97.1%-100%,respectively.The sequence alignment analysis showed the amino acid sequences of S1 gene existed deletion,mutation and insertion,and there existed variation among PEDV strains from Guangxi province.Analysis of the rate of evolution shows that the PEDV S gene has the fastest evolution rate.The phylogenetic trees based on S,M,N genes showed similar topology diagrams.The PEDV strains from Guangxi distributed in G?a,G?b,G?a and G?b subgroups,and most of them distributed in G?b and G?a subgroups.The results indicated that there existed genetic diversity of PEDV strains from Guangxi province and it should be reinforced for pathogen survailence and epidemiological investigation to provide basic data for effective prevention and control of PEDV.