Isolation And Identification Of Senecavirus A And Establishment Of RT-LAMP Detection Assay And Preparation Of Monoclonal Antibody

Senecavirus A(SVA)belongs to the genus Senecavirus of the Picornaviridae family,which is a single-stranded non-segmented RNA virus.SVA can cause blistering,ulceration and other symptoms in the nose and hoof of pigs.In severe cases,death will occur,which seriously harms the breeding industry.In this study we mainly collect a large amount of materials with lesion from a pig farm in Guangdong Province.The primers were designed base on the NCBI and firstly used to identify the illed materials with RT-PCR.The SVA were isolated on PK-15 cells,identified by real-time RT-PCR,indirect immunofluorescence and RT-PCR,and the isolates were assayed for half of the tissue culture infection dose(TCID₅₀).The results showed that after 3 of blind passage,the cytopathic effects(CPE)were abvious,which demonstrated successfully separation of SVA.The titer of TCID₅₀which was 10^-5.8of the isolate was determined.According to the SVA VP1 gene sequence published in Gen Bank,a pair of specific primers targeting VP1 gene was designed.The VP1 gene was amplified by PCR assay,then cloned into p MD-18T vector,and sequenced for homology and genetic phylogenetic tree analysis.The results showed that the amplified VP1 gene was 894 bp in length and the sequence alignment showed that the homology compared with other strain was92.3%?97.5%.The sequence of the amplified VP1 gene was the closest to the Guangdong isolates(KT321458.1,KX751946.1),and the homology was the highest.It is most distantly related to the American isolates(KU954086.1,KY618837.1)and the Thailand isolates(MF416219.1,KY368744.1).It can be found that the variability of the VP1 gene sequence was not high.In this study a reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay for SVA was also established.By downloading multiple sequences from NCBI and using Megalign alignment analysis,we chose conserved sequences and designed6 pairs of LAMP primers for real-time RT-LAMP assay and screening for suitable primers.After the conditions were explored and optimized in the early stage,the sensitivity of RT-PCR,real-time RT-PCR and real-time RT-LAMP were compared.The results showed that the optimal conditions were at 63°C for 60 min for amplification and the established detection method was specific.The limit of sensitivity was 100 copies per reaction for real-time RT-LAMP,which was higher than RT-PCR assay and real-time RT-PCR assay.The comparison results of 35 clinical samples showed that 16 samples were positive by real-time RT-LAMP assay,which was consistent with that by RT-PCR assay and real-time RT-PCR assay.We proved that the RT-LAMP can be used for application.In addition,the purified SVA VP1 protein was used to prepare monoclonal antibodies by immunizing BALB/c mice,the cells were fused with immunosuppressed mouse spleen cells and myeloma cells,the indirect ELISA method was used to screen positive hybridoma cells.Four positive hybridoma cells stably secreting antibodies were obtained by subcloning.The IFA results showed that all the four prepared monoclonal antibodies were able to react with SVA.The monoclonal antibody subtype analysis revealed#11,#14 and#33 for the heavy chain lg G1,#16 is the heavy chain lg G2a,and all the light chains are Kappa chains.The specificity was verified by vesicular stomatitis virus(VSV),swine vesicular disease virus(SVDV)and foot-and-mouth disease virus(FMDV).The results showed that the specificity was typical and the cross-reaction won't be occurred.The titer analysis#11 was 1:51200,#14 was 1:12800,#16 was 1:204800,and#33 was 1:6400respectively.The obtained four monoclonal antibodies can provide relevant basis and material basis for the study of VP1 structural function and the diagnosis of SVA.