Epidemiological Investigation,Isolation,Identification Of PDCoV And Establishment Of Real-time Fluorescent Quantitative PCR

Porcine deltacoronavirus Coronavirus(PDCo V)is a newly discovered coronavirus since 2012.Porcine deltacoronavirus Coronavirus is an enteric pathogen,and pigs of all ages are susceptible,but mainly cause diarrhea,vomiting and dehydration of piglets.C linical symptoms are similar to those of PEDV and TGEV but the mortality rate(30% to 40%)was lower than the latter two,often accompanied by a mixed infection with PEDV and TGEV.Infected pig pathology analysis reflect that the small intestine in the yellow liquid,intestinal wall thinning,transparent,histopathological showed a large number of small intestinal villi atrophy.Since PDCo V has occured,it has brought huge losses to the pig industry.Although PDCo V prevail largely,we do not infer the origin of PDCo V.Therefore,it is necessary for us to study the prevalence and sequence analysis of PDCo V,and establish a fast,stable,sensitive and accurate detection method have great significance.The virus titer is relatively low when porcine was early infected,so we established a nested PCR method successfully based on the N gene of PDCo V.The epidemiological examination of 420 pig diarrhea samples collected in Guangdong Province from 2012 to 2016 was carried out by nested PCR,the results of pathogenic test of 420 samples showed that the positive rate of PDCo V was 13.33%,and co-infection rate of PDCo V and PEDV was 5.95%.PDCo V-positive samples were inoculated with LLC-PK cells,this study successfully isolated a pig-type coronavirus,named PDCo V/CHGD/2016.The whole nucleotide sequence of PDCo V/CHGD/2016 was amplified,sequenced,then genetic analysis of the whole gene sequence and S protein sequence of the representative strain PDCo V/CHGD/2016 was carried out by using biological software.The whole genome of PDCo V/CHGD/2016 was 25419 bp in length.Whole genome analysis showed that PDCo V/CHGD/2016 shared the highest similarity(99.4%)with three Chinese strains (PDCo V/C HJXNI2/2015,C HN-JS-2014,CHN-HB-2014),and the lowest similarity(97.4%)with the two Thai strains.Spike gene analysis showed that PDCo V/CHGD/2016 had 95.9%-99.3% similarity with other Pecos.Among them,PDCo V/C HGD/2016 is close to PDCo V/CHJXNI2/2015,and distinct the Thai PDCo V strains.Compared with HK U-155 and CHN-AH-2004,PDCo V / CHGD / 2016 had three not(GTT)insertions in the 3 'untranslated region at 25047-25049.The phylogenetic trees based on whole genome sequence and Spike gene sequence showed that PDCo V/C HGD/2016 had the close phylogenetic relationship with Pecos,but it was far alway from bird deltacoronavirus.The small intestine of piglets infected with PDCo V was collected,then stained with HE to observe the histopathological changes,the results show that the main change was villi atrophy of small intestinal,which confirmed that PDCo V is a typical intestinal pathogens.The study above would provide some theoretical support and data analysis for the study of the virus in diagnosis,pathology,pathogenicity,epidemiology and molecular biology.In order to establish a rapid,sensitive,accurate and convenient method for the diagnosis and treatment,this study establish PDCo V SYBR Green ? Real-time Fluorescence Quantitative by Designing Primers based on the conserved gene N of PDCo V.The standard curve established show that threshold cycle has a good linear relationship with the template concentration The specific experiment showed that only PDCo V positive samples had fluorescence signal with the perfect S-type amplification curve appeared,while TGEV,PEDV,PRRSV,PRV,PCV and negative control were not amplified.The results show that the method of real-time fluorescence quantitative PCR established in this experiment is specific.In this study,the lowest copy number that can be detected by real-time fluorescence quantitative PC R was 81.4 copies / ul,which was 1000 times higher of than conventional RT-PCR.Repetitive experiment showed that the six S-type amplification curves were closely arranged,and the obtained C t values were bas ically the same and the variation was very small.It proved that the real-time PCR established in this experiment is good repeatability.C linical sample test show that the positive rate of real-time PCR was higher than that of conventional RT-PCR.This method was established successfully with the specificity was strong,the sensitivity was high,the reproducibility was good.In addition the quantitative analysis of the tested samples could save time and effort,which improve the detection efficiency and provide great convenience for the clinical diagnosis of PDCoV.