Establishment And Preliminary Application Of Fluorescent Quantitative RT-PCR Method And Indirect ELISA For Detection Of Porcine Deltacoronavirus N Gene

Porcine deltacoronavirus(PDCo V)is a newly discovered coronavirus mainly infected with suckling piglets.Clinical manifestations include acute watery diarrhea,vomiting,dehydration,and death.At present,the disease has a global distribution and has been detected in pig farms in many provinces in China.It has become a new type of enteric pathogen that harms the health of newborn piglets.In this study,N gene cloning and bioinformatics analysis of PDCo V Sichuan strain has been performed,fluorescence quantitative RT-PCR of PDCo V N gene was constructed,recombinant N protein was expressed,and the establishment of polyclonal antibody preparation and indirect ELISA was established,for the subsequent development of PDCo V Diagnostic and Prevention Research Materials.1.N gene cloning and bioinformatics analysis of PDCo VA pair of specific primers was designed based on the NCBI-registered PDCo V N gene sequence.The PDCo V N gene was amplified by RT-PCR from a fecal sample from a swine farm in Sichuan,and N gene cloning plasmid was constructed.The gene sequence analysis and mutation analysis were performed.The structure showed that the amplified N-gene of 1026 bp of PDCo V N gene and CH/Sichuan/S27/2012 was98.93%,and it was named CHN-SC2015 strain N gene.A genetic phylogenetic tree was constructed and the structure showed that the N gene of the CHN-SC2015 strain was closely related to the N gene of the PDCo V strain isolated from China mailand strain,and the closest relationship with the CHN/Jingsu/2014 strain.However,the PDCo V strains isolated from the United States,Vietnam,Japan,and Thailand are far from each other.The on-line software prediction of the N protein amino acid sequence of the CHN-SC2015 strain showed that the protein is a stable hydrophilic protein with abundant irregular curls,and there is no transmembrane region and signal peptide.2.Establishment of SYBR GREEN?fluorescence quantitative PCR method for the porcine delta coronavirusIn this study,a pair of specific primers were designed based on the conservedregions of the N gene of PDCo V CH/Sichuan/S27/2012 strain in the Gen Bank database to prepare a positive standard and a SYBR GREENI fluorescent quantitative PCR method for establishing a pig delta coronavirus was established.The results showed that the method has a good linear relationship in the range of 10⁴-10⁸,with a minimum detection of 10⁰ copies/?L of viral nucleic acid.Only the PDCo V shows an amplification curve.The rest of the viruses are negative,and intra-and inter-group variations The coefficient is less than 2.4%.Detection of 239 clinical samples,the positive rate was 5.43%.3.Prokaryotic expression of PDCo V N protein and preparation of polyclonal antibodiesThe p ET-28a(+)-N recombinant plasmid was constructed for expression.The size of the recombinant protein was approximately 44 k Da as determined by SDS-PAGE.The recombinant protein expression conditions were optimized.The results showed that the final concentration of IPTG was 0.8 m M at a temperature of30°C.The expression time was 3 h.Transetta strain carrying recombinant plasmid had the highest expression level.Recombinant protein could be expressed in supernatant and inclusion body,and the expression level in supernatant was significantly larger.Ni+affinity chromatography was used to select protein supernatant.After purification and protein concentration using ultrafiltration tubes,a recombinant protein with a concentration of 0.78 mg/m L was obtained.The immunized rabbits prepared a polyclonal antibody with a titer of 1:204800.4.Establishment and preliminary application of indirect ELISA for PDCo V N proteinBased on the purified N protein,an indirect ELISA for PDCo V was constructed.Through optimization of the ELISA reaction conditions,the antigen coating concentration was 1.0?g/m L,the serum dilution was 1:100,and the enzyme-labeled secondary antibody dilution was 1:4000.The detection effect was the best;the determination standard was OD₄₅₀?0.201.There was no cross reaction with positive serums such as PEDV,PRSSV,JEV,HPS;its sensitivity could reach positive serum with 1600 times dilution;in the repeatability test,both intra-and inter-assay coefficients of variation were less than 10%.Using this method to detect 387 serum samples collected in Sichuan,the positive rate was 0.26%.The indirect ELISA method established in this study has the characteristics of strong specificity,simplicity,and rapidity.It can be used for the rapid detection of PDCo V antigen,and further confirms the feasibility of N protein as an antigen.