Establishment And Application Of RT-PCR,RT-LAMP And Antibody-capture Indirect-ELISA Methods For Detection Porcine Deltacoronavirus

Porcine Deltacoronavirus is a newly discovered coronavirus that can cause pigs,especially in newborn piglets diarrhea,high incidence and mortality rate,which is one of the most important causes of neonatal piglets mortality.Porcine Deltacoronavirus belongs to the genus Coronavirus,family coronaviridae.PDCoV has reported in 2010 and expanding to the United States,China and so on.At present,the pathogenesis and replication mechanism of PDCoV is not clear,only to understand the characteristics of piglets caused by swine epidemic diarrhea virus similar.In view of the widespread epidemiology and potential threat of PDCoV in swine,this study established RT-PCR,RT-LAMP and indirect ELISA for PDCoV antibodies.1.Establishment of PDCoV RT-PCR and its epidemiological investigationBased on the result of multialignment analysis on the published PDCoV genome sequences on GenBank database,a pair of primers specific to the conserved nucleocapsid(N)gene of PDCoV was designed by using the online software Primer 3.0,and then a RT-PCR for detection of PDCoV was established by utilizing the designed primers.A single expected DNA fragment band of 329 bp in size was amplified by using the designed primers.Sequences of the amplicons selected were subjected to a BLAST search against the Gen Bank database,and the results indicated that the sequences hit PDCoV with a nucleotide identity as high as 99.1%,demonstrating that the amplified targets were PDCoV.A phylogenetic tree was generated based on eight partial N gene of PDCoV obtained in this study,18 reference PDCoVs from other countries/areas,and two representative PEDV and two selected TGEV sequences with corresponding fragments.All of the PDCoV strains were classified into a cluster,distinct from PEDV and TGEV strains.The eight PDCoV sequences obtained in this study showed that the highest similarity with a Korea PDCoV strain KNU14-04,but less similarity to two Hong Kong strains HKU 15-44 and HKU 15-155.No cross amplification for PEDV,TGEV,PKoV,PAstV,PRRSV and CSFV was observed,indicating the established RT-PCR assay was highly specific.The detection limit of 1.0×103 copies/?L of the assay was determined based on the templates of the recombinant plasmid with 10-fold serial dilution(from 1.0×106 to 1.0×101 copies/?L),indicating the assay was sensitive.A total of 249 diarrheal fecal/intestinal samples of sows and piglets collected in Jiangxi Province,China from 2012 to 2015 were examined by the established assay,and the positive rate of PDCoV was 31.3%(78/249),and the samples from sows showed somewhat lower positive rate(27.8%)when compared with that from piglets(31.9%).PDCoV could be detected in samples as early as 2012.2.PDCoV RT-LAMP detection method was establishedAccording to the results of the comparison,the total length of PDCoV N gene was selected as the target sequence,and two sets of RT-LAMP primers were designed.PDCoV RT-LAMP was established by using the total RNA extracted from PDCoV positive pathogen as template.After optimization of the reaction conditions,the results show that the optimum conditions of the reaction are 63 ? constant temperature water bath 70 min,and the method has high sensitivity and good specificity.RT-PCR,RT-PCR and nested RT-PCR were used to detect 192 clinical samples.The results showed that the sensitivity of RT-LAMP was 100 times that of RT-PCR,which was similar to that of nested RT-PCR.3.Expression of the prokaryotic expression of the N geneThe full-length cDNA of PDCoV N gene was amplified by total RNA extracted from PDCoV positive pathogen.The amplified fragment was ligated with expression vector pcold ? to construct recombinant expression plasmid p Cold-PDCoV-N and was transformed into the BL21(DE3)competent cells,and the recombinant bacterial OD600 nm value was 04-0.6 in the LB broth.After 30 min of ice bath,the final solution was 0.8 mM IPTG and the expression was 21 h at 15 ?.The recombinant protein was about 41 kDa,The protein is mainly present in a soluble form.Western Blot test was performed using PDCoV positive serum as primary antibody,and the results showed that the immunogenicity was good.4.Establishment of PDCoV indirect ELISA assayThe purified antigen of PDCoV N was used as the coating antigen,and the optimal antigenic coating solubility and antibody were determined by checkerboard titration.The anti-bacterial dilution factor,different blocking fluid,blocking time,primary reaction time and secondary action time The results showed that the amount of antigen coated was 5 ?g,3% calf serum was closed for 2 h,the serum was diluted 100 times,the reaction was 45 min,the enzyme labeled secondary antibody was diluted 4000 times,The reaction was optimized for 1 h.Determined by statistical analysis of critical value was 0.316,and there was no cross-reaction with six positive pigs of porcine epidemic diarrhea virus and had good specificity.The coefficient of variation of the intermittent and intermittent repeat tests was less than 10%,and the reproducibility and stability were good.The positive rate was 12.8%(36/282),which indicated that PDCoV was prevalent in the pig herds in our province.In this study,PDCoV antibody capture indirect ELISA was used to provide experimental evidence for clinical monitoring and epidemiological investigation of PDCoV infection in swine.