Application Of Molecular Differential Diagnostic Method For SVA And Prokaryotic Expression Of Viral Protein VP1

The Senecavirus A(SVA)is the only representative of the family Picornaviridae,species Senecavirus.Since it was first discovered in Canada in 2007,it has appeared around the world.In 2015,the first case of SVA infection was found in Guangdong Province,China.It was characterized by fever,claudication,hoof and nasal blisters which was similar to the foot-and-mouth disease.There are no commercially available vaccines and diagnostic kits on the market,and establishing a rapid and accurate differential diagnosis method is critical for the prevention and control of the disease.In this study,the dual RT-PCR and SVA indirect ELISA method were established to identify the SVA and the FMDV.In this study,two pairs of specific primers were designed based on the conserved gene sequences of SVA and FMDV,and a duplex RT-PCR method was developed to detect SVA and FMDV simultaneously.Then we optimized reaction conditions and system,the best annealing temperature was 56.1°C,the optimal primer concentration was 0.2?M,the best 10×PCR buffer concentration was 1 mM,the best Taq DNA polymerase concentration was 2.5 U.Further specificity and sensitivity analysis showed that were there no positive amplification for PEDV,SVDV,VSV,PRRSV,PCV,CSFV,and the minimum detection amount of SVA and FMDV were 1.40×10~2 copies/?L,1.10×10~2 copies/?L,indicating that the method were highly specificity and sensitivity.The 110 clinical samples were detected by this method.The positive detection rate of SVA was 16.3%,the positive detection rate of FMDV was 1.8%,and the mixed infection rate was 1.8%,the results were consistent with single RT-PCR.Therefore,the duplex RT-PCR method,which can detect both SVA and FMDV,provides a good method for the differential diagnosis of the blisters and laid a foundation for the development of diagnostic kit.A pair of specific primers were designed according to the sequence of SVA HN16strain(GenBank:MF893200)VP1 gene isolated in our laboratory,while BamH I enzyme cutting sites and Xho I enzyme cutting sites were added to the downstream and upstream primers respectively.The VP1 gene was amplified from the positive material and connected to the pET-32a expression vector.Then the recombinant plasmid pET-32a-VP1was successfully constructed,preliminary identification of recombinant fusion protein by SDS-PAGE is 52 kD,mainly exists in the form of inclusion body.Furthermore,IPTG concentration and induction time were optimized and screened,the best IPTG concentration was 1 mM and the optimal induction time was 6 h.The recombinant protein was prepared by the best inducement condition,and Western blot was carried out on the purified recombinant protein.The results showed that the expressed target protein could react with SVA positive serum,which laid the foundation for the subsequent indirect ELISA method.The purified,refolded recombinant protein was used as envelope antigen of indirect ELISA and to optimize conditions.The optimum antigen inclusion concentration was 5.5?g/mL,the best sealing fluid was 3%BSA,the best closing time was 37°C 2 h,the best serum dilution ratio was 1:100,the best HRP-conjugated antibody was 1:5000 and the best substrate color reaction time was 37°C 15 min.The critical value of the negative serum was 0.206 by statistical method,and the specificity and repeatability test were conducted showing highly specificity and repeatability in the batch.The established indirect ELISA method of SVA VP1 protein is feasible,which laid a foundation for the development of SVA indirect ELISA diagnostic kit.