Establishment And Application Of An Indirect ELISA Method For Antibody Detection Of Novel Goose Parvovirus

Since the second half of 2014,plenty of duck breeding flocks in Anhui,Jiangsu,and Shandong province broke out an infectious disease called short beak and dwarfism syndrome?SBDS?.The disease was characterized with beak atrophy,tongue exposure,and growth retardation.Farmers named it“big tongue disease”.Researchers demonstrated that the pathogen of SBDS disease was a novel goose parvovirus?NGPV?after virus isolation and animal reproduction experiment.NGPV showed over 95%nucleotide similarity with goose parvovirus?GPV?and the two viruses located in the same evolutionary branch.In the present study,we developed an indirect ELISA method used to detect NGPV antibody in duck serum,to provide a reliable detection method for the serology diagnosis of NGPV.By using this method,we investigated duck serum samples from different regions in Shandong province,which facilitated the serology data of NGPV.The research contents were as follows:The prokaryotic expression and purification of capsid protein of NGPV:According to the gene of capsid protein in Genbank,we designed two pairs of primers to amplify VP1 and VP3.Virus DNA of NGPV was abstract in NGPV positive samples,which was used as template for PCR.Then VP1 and VP3 genes were ligated to the pET-32a?+?vector to construct recombinant plasmids pETVP1 and pETVP3.Next,the two recombinant plasmids were transformed into E.coli BL21?DE3?bacteria to express protein.The crude protein was purified by using urea gradient method and tested concentration by BCA Testing Kit.The concentration of VP1 protein and VP3 protein was 2.18?g/?L and 3.7?g/?L,respectively.Western blot analysis showed that the VP1 and VP3 protein can specifically react with NGPV positive serum,while antibody of other pathogens and serum of healthy duck cannot react with it.The development of an indirect ELISA method for the detection of NGPV antibody in duck serum:VP1 and VP3 protein was used as the coating antigen of ELISA method to test NGPV positive serum and negative serum under the same testing condition.The results showed that the P/N value of ELISA using VP3 protein as coating antigen was much higher than ELISA using VP1 protein as coating antigen.Therefore,the VP3 protein was selected as the antigen to establish the indirect ELISA method.After the optimization of a series of conditions for ELISA method,we found out the optimal conditions.The concentration of coating antigen?VP3 protein?was 2?g/mL;The ELISA method used 5%non-fat dry milk as sealing fluid?250?L/well?and sealed for 2 hours;The serum samples were diluted by 400-fold?100?L/well?and incubated at 37°C for 1.5 h;Goat anti-duck antibody was diluted at 4000-fold?100?L/well?and blocked at 37°C for 2 h;TMB solution?100?L/well?was acted at 37°C for 10 minutes.A total of 36 NGPV negative serum were tested using this indirect ELISA method,the critical value was calculated based on statistical principal.When OD₄₅₀50 value of samples was over 0.2024,the samples were considered positive for NGPV;while the OD₄₅₀50 of samples was below 0.183,the samples were considered negative for NGPV.Specificity test of the ELISA method showed that antigen cannot react with the antibody of other pathogens including DHV,AIV and DPV.The repetitive test showed that the indirect ELISA method was with good repetitive feature.A total of 120 serum samples were tested by the indirect ELISA in comparison with virus-neutralization test?VN?.The total coincidence of the two methods was 85.8%.Amplification of the indirect ELISA method:In this study,a total of 970 serum samples were collected from five duck flocks in Shandong province.The results showed that the positive rate of NGPV in flock level was 100%.The positive rate in individual level was ranged from 17.6%-62.6%,with the highest detection rate?62.6%?in Weifang.The detection rate of two flocks in Tai'an city were much lower,including Xintai flock?17.6%?and Feicheng flock?26.0%?.The NGPV positive rate on average was 39.7%.