Establishment And Application Of Antibody Detection Method For Porcine Sapelovirus 1

Porcine sapelovirus 1(PSV-1)is a single-stranded,positive-sense RNA virus,belonging to the genus Sapelovirus,within the family Picornaviridae.PSV-1 infections occur worldwide and are associated with a wide spectrum of symptoms ranging from asymptomatic to clinical manifestations including diarrhea,pneumonia,polioencephalomyelitis,and reproductive disorders.In this study,an indirect ELISA method for the detection of PSV-1 antibodies was established,and compared with the established indirect immunofluorescence technique(IFA).The contents are as follows:1.The IFA method for detecting PSV-1 antibody was established by using PSV HuN1/2016 strain isolated by our laboratory.The primary antibody and fluorescent secondary antibody in the method were diluted 300 times.2.The recombinant plasmid was constructed by introducing PSV-1 encoding VP1 gene into pET-28a(+)vector.Western blot analysis showed that the purified recombinant inclusion bodies had good specificity and reactivity.The indirect ELISA method established by VP1 was as follows: the antigen was coated with 250 ng / well;the serum was diluted 100 times;the serum was incubated at 37 ? for 30min;the enzyme labeled secondary antibody was diluted 6000 times and was incubated at 37 ? for 30min;the substrate was incubated at 37 ? for 20 min.We define the positive,if the S / P value ? 0.235,S / P value ? 0.191 value is negative,and the remaining was suspicious.The total coincidence rate was 82.6% between the established ELISA method and IFA method.Coefficient of variation of intra and inter antigen plate batch were 1.6 to 7.0%.3.We detected 400 serum samples collected from 5 intensive pig farms,The results showed that dynamic changes of PSV-1 antibody in the five fields were similar: the antibody of 2-6 weeks old piglets were low and showed a descending trend;the antibody of 8-12 weeks old piglets showed an ascending trend;until the 18 weeks old piglets,the average antibody level was basically stable;the average of antibody was low in sow group.