Establishment Of Real-time Quantitative PCR Method For Porcine Deltacoronavirus And Its Genetic Evolution Analysis

Porcine deltacoronavirus(PDCoV)causes the clinical symptoms similar to porcine epidemic diarrhea virus(PEDV),which will easily confuse the clinical diagnosis.Therefore,the aim of this study was to establish a real-time fluorescence quantitative PCR method for PDCoV detection,which can provide an effective technical method for clinical differential diagnosis.Furthermore,to enrich the gene characteristics and genetic information database of PDCoV in domestic,the whole genome of a PDCoV isolate was sequenced and its genetic variation characteristics was analyzed widely,providing scientific data reference for the prevention and control of the disease.1.Establishment and application of a real-time PCR method against PDCoVIn view of the high conservation of PDCoV N gene among coronaviruses,a fragment with highest conservative coefficient was screened by alignment of the N gene of PDCoV strains prevalented over the world.Then a pair of specific PCR primers were designed.After PCR amplification,the target fragment against PDCoV N gene was cloned into T vector.Finally,the positive standard plasmid p EASY-N was obtained.The concentration of the standard plasmid was tested followed by 10-fold serial dilution,which were then used as the template for construction of the standard curve by real-time PCR.The optimized reaction conditions were as follows:95?,30s;95?5s,58?34s,40 cycles;95?15s;60?1min;95?15s.And the optimized primer concentration was 1.2?mol.The results showed that the standard curve had the linear relationship with the slope of-3.232 and the correlation coefficient of 0.999.And the melting curve was good which confirmed that the real-time PCR primers had good specificity.The sensitivity of this method was good with the minimum detection limit of 5.83×10~1copies/?.And the specific test showed that PEDV,TGEV,PRV and other non-specific viruses could not be detected.Finally,60 feacal samples collected from the pig herds around Shanghai were detected using the PDCoV real-time PCR method developed in this study.The results showed that the PDCoV positive detection rate was 18.3%,higher than that of conventional PCR(6.7%).In conclusion,this study successfully established a rapid and sensitive pathogen detection method for PDCoV,providing a measure for clinical continuous monitoring of PDCoV and timely warning and prevention.2.Whole-genome sequencing and genetic evolution analysis of PDCoVAccording to the genome of the PDCoV strain CHN-HB-2014(Gen Bank No:KP757891.1),16 pairs of specific primers were designed and optimized,respectively.Then,the full-length sequence of a PDCoV isolate SH316 was segmented amplified and spliced using DNAstar software.The results showed that the complete geome length of SH316 was 25424nt.Homology comparation and phylogenetic tree indicated that this isolate had the highest homology with CHN-GD16-05 and CHzmd2019,which were clustered in the same branch with the America PDCoV strains while had far gentic distance with the Thai and Vietnamese strains.The recombination analysis revealed that there is genetic recombination existed among SH316,HKU15-44,and CH/SXD1/2015 with the a potential recombination breakpoint at 2258-2884bp.In addition,there were amino acid site mutations in S gene,suggesting this isolate was different from the domestic PDCoV epidemic strain,which laid a foundation for further research on the antigenicity of PDCoV.