Isolation,Identification Of Senecavirus A Virus And Establishment Of Detection Methods

Senecavirus A(SVA),as an exotic pathogen that can cause vesicular symptoms in pigs,has caused serious economic losses to pig farming worldwide.In recent years,many scholars at home and abroad have investigated the infection and epidemiology of SVA in China,and the results show that SVA infection in China is relatively serious and has an upward trend,and it is extremely important to avoid huge losses caused by large-scale SVA infection in pigs.As a region with a high scale of pig breeding in western China,Chongqing urgently needs to conduct in-depth investigation of SVA infection in local large-scale pig farms and establish SVA detection methods for pigs.Test1.Isolation and identification of a strain of Senecavirus A in ChongqingIn order to understand the prevalence of SVA in large-scale pig farms in Chongqing in recent years and master the epidemic strains of SVA,the disease materials suspected of SVA infection in large-scale pig farms in Chongqing from 2021 to 2022 were isolated and identified,and whole genome sequencing was performed,and 25 representative strains at home and abroad were selected for homology comparison and evolutionary tree according to ORF region.The results showed that one strain of SVA was successfully isolated and named SVA/CH-CQ(Gen Bank:OP696593).Through the comparison of homology with representative strains at home and abroad,it was found that SVA was different from the circulating strains at home and abroad.Among them,SVA/CH-CQ had the highest homology with HS-01 strains,accounting for 99.95%.The lowest homology with SVV-001 was 93.51%;Phylogenetic tree analysis showed that CH-CQ strain was the closest related to HS-01 strain and most distant to IN＿Purdue＿4914-26 and GO3 strain.The results showed that there was an epidemic of SVA in large-scale pig farms in Chongqing,and there was evolution and variation of SVA during the epidemic process,and corresponding measures should be taken for comprehensive prevention and control of the disease.Test2 Prokaryotic expression of VP2 protein of Senecavirus A and establishment of indirect ELISA detection methodIn order to establish an indirect ELISA detection method for Senecavirus A(SVA),specific primers were designed according to the VP2 gene of the isolated SVA/CH-CQ strain,amplification fragments and p ET32a(+)vectors are double-digested,then ligated and identified,and the optimal expression conditions were found by optimizing the protein expression temperature,time and IPTG concentration using the E.coli prokaryotic expression system,and the inclusion body protein activity was restored by urea denaturation recombination.After being correctly identified by SDS-PAGE and Western-blot,it was used as ELISA-coated antigen.Optimize the square matrix of various factors that affect ELISA testing,test for specificity,sensitivity,and repeatability,and validate the ELISA test results using Western blot.The results showed that the p ET32a(+)-VP2 was successfully constructed after restriction endonuclease digestion and sequencing validation.The optimal expression conditions for SVA VP2 protein were IPTG 0.6 m M,30℃,and 6 hours.After protein refolding,it was verified by SDS-PEGE and Western blot that it could be used as an ELISA coating liquid.The optimal expression conditions of SVA VP2 protein were IPTG 0.6 m M,30℃,6 h.Recombinant proteins has been verified by SDS-PEGE and Western-blot for use as ELISA coating solution.The optimized conditions of ELISA square matrix were as follows:1μg/m L antigen coating overnight at 4℃,5%BSA blocking time for 1 h,1:500 dilution primary antibody incubation for 2 h,1:5 000 dilution secondary antibody incubation for 1 h;The yin and yang differences of this method were significant,and the specificity,sensitivity and repeatability were good,which were consistent with the Western-blot verification results.The results show that an expression system for SVA VP2 protein is constructed,and an indirect ELISA detection method for SVA antibody with good specificity,sensitivity and reproducibility is established by using the expressed VP2 protein,which provides an effective tool for SVA prevention and control and clinical detection.Test3 Establishment of SYBR GreenⅠPCR detection method for Senecavirus AIn order to establish the SVA Real-Time PCR detection method,this study designed specific primers according to the isolated SVA/CH-CQ strain VP2 gene,amplification fragments and p MD-19T vectors are double-digested,and used the standard plasmid to be diluted for Real-Time PCR and plotted the standard curve,through the optimization of the reaction procedure and system,the SVA Real-Time PCR detection method was established and compared with the ordinary PCR method.The results showed that the p MD-19T-VP2 plasmid was successfully constructed,and the Ct value of the PCR detection method established in this study had a good linear relationship with the standard template in the range of 1.0×10~2～1.0×10~8 copies/μL,and the correlation coefficient was0.9988.The method was used to detect samples of common diseases such as PEDV and TGEV without amplification,indicating that its specificity was good.The lower detection limit of this method can reach 1.0×10~2 copies/μL,which is 100 times higher than that of conventional RT-PCR.The coefficient of variation is always less than 2%,indicating that the test is reproducible.The results show that the SYBR Green I.PCR method established in this study can be used as an important diagnostic technique for SVA,which can provide help for the rapid clinical detection of SVA and facilitate the prevention and treatment of SVA.