Development Of Antibody Indirect ELISA And Genotyping QRT-PCR Detection Method For Porcine Rotavirus

Porcine rotavirus（Po RV）is an important pathogen causing outbreaks of diarrhea in pig farms,and it has numerous genotypes with weak cross-protection between different genotypes.Clinically,co-infection with multiple genotypes can be seen,exacerbating piglet diarrhoea and even leading to death.Po RV strains can recombine with strains of other species,resulting in interspecific transmission and posing a great threat to public health safety.In order to better monitor and understand the infection of Po RV,Po RV fecal samples collected from three large pig farms were isolated and characterized in this trial,and the sequence analysis of the isolated Po RV VP7 gene was performed.Meanwhile,the VP8protein of Po RV G4P[23]strain was used as the detection antigen to establish an indirect ELISA.Finally,a multiplex q PCR method was established in view of the high detection rate of G4,G5 and G9 Po RV.The specific research results are as follows:1.Isolation and identification of Po RVTen positive fecal samples of Po RV were selected to inoculate MA104 cells,and the typical rotavirus lesions appeared after four generations.After IFA detection and RT-PCR identification,five strains of Po RV were finally isolated,named Po RV-9,Po RV-32,Po RV-97,Po RV-12 and Po RV-38.The VP7 gene of each isolate was sequenced and analyzed,Po RV-9 and Po RV-38 were identified as G4 Po RV,and the strain with the highest homology was porcine HLJ/15/1 isolated from china,with 95.8%homology;Po RV-12 and Po RV-32were G5 Po RV,and the strain with the highest homology was porcine DZ-2 isolated from china,with 96.1%homology;Po RV-97 was G26 Po RV,and the strain with the highest homology was porcine TJ4-1 isolated from Japanese,with 95%homology.2.Establishment of an indirect ELISA for detection of antibodies against Po RVThe recombinant VP8 protein of Po RV was expressed in E.coli,then recovered by SDS-PAGE and used as coating antigen to establish an indirect ELISA method.The reaction conditions were optimized as follows:2μg/m L antigen was coated for 1 h at 37℃;2.5%BSA blocked at 37℃for 2.5 h;primary antibody diluted to 1:80 and incubated at37°C for 1 h;the second antibody was diluted to 1:20000 and reacted at 37℃for 1 h;the reaction time of TMB was 15 min at room temperature in the dark.23 negative serum samples of Po RV antibodies were detected by the established ELISA method,and the determination criteria were established:when the OD₄₅₀of the tested sample>0.438,it was considered as positive;When the tested sample OD₄₅₀<0.380,it was judged as negative.When the result of the tested sample was in between,it was judged suspicious.Antibody-positive Serum samples of PRRSV,CSFV,PCV2,PEDV and PRV were tested by this method.The results were all negative and no cross reaction occurred.When the Po RV antibody positive serum was diluted 1:1280,the method could still be judged as positive,indicating that the method had good sensitivity.The intra-assay and inter-assay coefficients of variation were within 10%,which means that the method has good repeatability.3.Establishment of a multiplex fluorescent quantitative PCR for detection of Po RV G4,G5 and G9 genotypeThree pairs of primers and probes were designed for the VP7 genes of G4,G5 and G9Po RV,and the conditions were optimised to establish a multiplex fluorescent quantitative PCR assay to differentiate between these three genotypes of Po RV.The results of the specificity test showed that the method was able to specifically detect the corresponding genotypes under both single and multiplex conditions,indicating that the specificity of the method was good.The sensitivity test showed a minimum detection limit of 10³copies/μL for all three genotypes,indicating that the method is sensitive.Repeatability test results showed that the coefficient of variation within the group was within 2%and the coefficient of variation between the groups was within 3%,indicating that the method has good reproducibility.Fifty-five clinical diarrhoea samples were tested by the developed method.14 samples of G4 genotype,6 samples of G5 genotype and 8 samples of G9 genotype were detected,of which 3 samples were detected as mixed infections.12 samples of nucleic acid were selected for sequencing and compared with the results of the self built method.11samples showed identical results,while 1 sample showed mixed infection using the self built method.