Establishment Of PDCoV And TGEV Dual Fluorescence Quantitative PCR And Identification Of Their Biological Characteristics

Porcine deltacoronavirus（PDCoV）and Transmissible gastroenteritis virus（TGEV）are one of the causes of acute diarrhea,vomiting,dehydration,and even exhaustion in piglets less than 2 weeks old.At present,as a new coronavirus,PDCoV still lacks effective vaccines for prevention and control.The intestinal problems of piglets caused by mixed infection of TGEV and other enteroviruses should not be underestimated.In addition,the two can cause piglets to appear similar clinical symptoms and pathological characteristics,causing great distress to the clinical differential diagnosis.Therefore,the establishment of rapid and accurate differential diagnosis methods and the development of effective vaccines have become the current focus of disease prevention and control.In order to establish a rapid,efficient,specific and sensitive diagnostic method,this study designed and synthesized specific primers and Taq MAN probes according to the conservative regions of the N gene of PDCoV and TGEV,and established a dual fluorescence quantitative PCR detection method for PDCoV and TGEV.The results show that there is a good linear relationship between the cycle threshold of this method and the logarithm of plasmid copy number,R²_（P）=0.9994 and R²_（T）=0.9969.It has no cross-reactivity with PEDV,PRV,PRRSV,CSFV and RV,and has strong specificity.The minimum detection limits for PDCoV and TGEV are 2 copies/μL and20 copies/μL,respectively,which are 1000 times and 100 times higher than PCR sensitivity.The intra-assay and inter-assay coefficients of variation of PDCoV and TGEV are both less than 2%,which has good repeatability.The detection of clinical samples showed that the positive rates of this method for PDCoV and TGEV were5.6%（6/114）and 8.8%（10/114）,respectively.The detection rate of mixed infection was 4.6%（5/114）,which was higher than that of conventional RT-PCR has a higher detection rate.The results show that the dual fluorescence quantitative PCR established in this study is strong,sensitive,reproducible and stable,and can provide technical support for monitoring pathogen infection and studying epidemic laws.In order to grasp the biological characteristics of PDCoV CH7328 strain and TGEV CH8438 strain,the three aspects of virus growth characteristics,pathogenicity and full gene sequence sequencing and comparison analysis were explored.According to the growth curve,PDCoV CH7328 and TGEV CH8438 both proliferated at 6 h,and reached their peak titer at 36 h and 24 h,respectively.The corresponding TCID₅₀was 10^-8.0/m L and 10^-6.3/m L,and then gradually decline.Pathogenicity tests found that the small intestines of challenged piglets were thinned and transparent and mesenteric congestion,and the intestinal villi cells of the jejunum appeared necrosis,shedding and reduction of intestinal villi.By sequencing the entire gene sequence of the virus strain,the genome lengths of PDCoV CH7328 strain and TGEV CH8438 strain were25420 bp and 28488 bp,respectively.Sequence comparison analysis showed that PDCoV CH7328 strain is in the same evolutionary branch with HKU15-155,CHN-HN-2014 and GD strains and has high homology.The amino acid sequence of S protein has a greater degree of variation,and the amino acid sequence of N protein is relatively conservative.TGEV CH8438 strain is in the same branch with H16,JS2012,attenuated H,TS and TGEV Miller M60 strains,with high homology,and the amino acid sequences of S and N proteins are relatively conservative.These studies provide an experimental basis for the screening and prevention of effective antigens for PDCoV infection and TGE in the future.