Isolation,Identification And Establishment Of Fluorescent Quantitative PCR Detection Method For Porcine Deltacoronavirus

Porcine Deltacoronavirus?PDCoV?is a pig intestine coronavirus that causes severe watery diarrhea,vomiting,and dehydration in pigs.The PDCoV genome is a single-stranded positive-stranded RNA with a similar structure to the PEDV genome.The genome size is 25.4 kb and contains 5'and 3'untranslated regions and seven open reading frames.With the continuous epidemic and evolution of PDCoV in the world,it has brought new challenges to the prevention and control of diarrheal diseases in pigs.Therefore,extensive collection and identification of PDCoV epidemic strains and the establishment of appropriate rapid diagnostic methods are important for the effective prevention and control of PDCoV.In this study,pig diarrhea samples from various provinces in China were collected,and virus isolation and identification of PDCoV-positive diarrhea samples were conducted.The materials and foundations were accumulated to fully understand the prevalence of PDCoV in China and to develop new and effective prevention and control products.In addition,a rapid PDCoV detection method based on TaqMan real-time fluorescence quantitative PCR was established to provide an effective and accurate method for rapid clinical diagnosis of PDCoV.?1?Isolation and identification of porcine delta coronavirus:During the period from 2016 to 2017,608 samples of diarrhea diarrhea were collected from 9 domestic provinces including Henan,Xinjiang and Heilongjiang.The samples were detected by RT-PCR and sequenced,and a cell-adapted strain CH/HNZZ27/2016 was successfully isolated by in vitro adaptability of 8 PDCo V-positive samples on LLC-PK1 cells.The strains were purified by plaque assay and successfully transferred to the 30th generation(P_1-30).Passaging viruses were identified by PCR,immunofluorescence assay?IFA?,and electron microscopy.The cell cultures of P₅,P₁₀,P₁₅,P₂₀,P₂₅,and P₃₀ were all amplified by PCR and were confirmed to be PDCoV by sequencing.The result of immunofluorescence detection showed that P₃₀ could detect PDCoV antigen with green fluorescence after 24 hours.Physicochemical analysis showed that the strain was sensitive to heat,insensitive to chloroform,and easily inactivated in the pH3 environment.Virus titer assay results showed that half of the P₃₀ tissue infective dose(TCID₅₀)was 10^-6.46.4 TCID₅₀/0.1 mL.The nucleotide and amino acid sequence analysis of the S gene revealed that the S gene of this strain was related to CH-01 and CHJXN12 strains in China,and HKU15-144,HKU15-155 in Hong Kong,and lllinois133,lllinois134,IOWA136,and Minnesota140 in the United States.It is in the same evolutionary branch as the Ohio 137 strain;it is not in the same evolutionary branch as the S5022,S5024 in Thailand.?2?Establishment of a Rapid Detection Method for TaqMan Fluorescent Quantitative PCR for Porcine Deltacoronavirus:Using the published PDCoV N gene sequence to design and synthesize specific primers and TaqMan probes,a plasmid standard was constructed,and a TaqMan real-time fluorescence quantitative PCR method for detecting PDCoV was established using the standard as a template,and the method was sensitive and specific.Sex and repeatability were evaluated.The results showed that no fluorescence signal was detected when porcine epidemic diarrhea virus,swine kub virus,porcine reproductive and respiratory syndrome virus,and foot-and-mouth disease virus were used as templates,indicating that the method has good specificity Replicate experiments were performed with plasmid plasmids of2.66×10⁶,2.66×10⁵,and 2.66×10⁴ copies/?L.The coefficient of variation of the cycle threshold Ct was less than 2%,indicating that the method has good specificity;The slope of the standard curve is-3.461,and the correlation coefficient is R²=0.998,indicating a good linear relationship between the threshold and the template concentration.A 10-fold serially diluted plasmid standard,with a minimum detectable plasmid DNA of 2.66×10¹ copies/?L,indicates that this method has good sensitivity.Using this method,194 samples of clinical swine feces samples were tested.The results showed that the positive rate of PDCoV was 22.1%,which was significantly higher than 11.9%of conventional RT-PCR,indicating that this method can be applied to clinical diagnosis and quantitative detection of PDCoV.