Establishment And Application Of Epidemiological Investigation And Fluorescent Quantitative PCR Detection Method For Porcine Delta Coronavirus

Porcine deltacoronavirus(PDCoV)is a newly discovered intestinal coronavirus that is clinically characterized by watery diarrhea,vomiting,rapid dehydration,and failure.Pigs can be infected at all stages,and newborn suckling pigs are most susceptible to infection,with an incidence rate of 50% to 100% and a mortality rate of 100%,causing huge economic losses to the pig industry.There are currently no measures available for the treatment and prevention of PDCoV infection.In order to reveal the genetic variation and epidemic law of PDCoV,this study conducted PDCoV detection on pig diarrhea samples in Shandong and Guangxi,and obtained a full length of PDCoV.At the same time,this study established a rapid and efficient PDCoV fluorescence quantitative method diagnosis.All work provide more comprehensive theoretical basis for the prevention and control of PDCoV.The research was carried out as follows:1.Based on the PDCoV,TGEV PEDV and PoRV gene sequences published by GenBank,primers were designed and tested.From September 2017 to January 2019,140 samples of piglet diarrhea from large-scale pig farms in Shandong and Guangxi were collected for testing.The results showed that 13 of the 140 doses of PDCoV were positive for PDCoV nucleic acid,the positive rate was 9.29 %;the positive rates of PEDV,TGEV and PoRV were 20%,29% and 13.75%,respectively,among which PEDV and PDCoV,PoRV and PDCoV The mixed positive rates were 6.43% and 3.57%,respectively.It indicates that PDCoV is easy to mix infection with other viruses.According to the gene sequence of PDCoV CHN-AH-2004 strain published in GenBank,25 pairs of specific primers were designed to perform RT-PCR positive RT-PCR fragmentation amplification,and the full length of one PDCoV genome was obtained by splicing.Sequence analysis homology and phylogenetic tree analysis of PDCoV epidemic strains and reference strains were performed using biological information software.The results showed that the genome of PDCoV CHN-GX-2018 was 25,407 bp in length,and there were two major changes compared with the US isolates:(1)There are two amino acid deletions in ORF1a/1b(400-403 aa,756-759 aa position);(2)1 amino acid deletion in S gene.Homology analysis showed that genetic analysis of whole genome and S gene showed that PDCoV CHN-GX-2018 has high homology with CHN-AH-2004.According to the phylogenetic tree analysis,PDCoV CHN-GX-2018 is in the same branch as the reference strain in Southeast Asia,and its affinity is relatively close.2.A rapid detection PCR method for PDCoV was established.A specific primer was designed based on the PDCoV N gene sequence,and a real-time PCR method was established.The results show that by linear regression analysis,the slope of the standard curve was-4.90 and the correlation coefficient was 0.999,indicating a good linear relationship between Ct and template concentration.Specific melting peaks appeared in all dilution standard templates;the lower limit of detection was 6.31×101 copies/?L;the specificity showed that PEDV and TGEV were not amplified;60 clinical pig stool samples were tested by this method.The positive rate of PDCoV was 5.0%,indicating that the method established in this study can detect PDCoV quickly and sensitively,which lays a foundation for the prevention and vaccine research of PDCoV.