Development Of Q-PCR And Indirect ELISA For The Detection Of Bovine Parainfluenza Virus Type 3

Bovine parainfluenza virus type 3(BPIV3)is a single and negative strand RNA virus,which is the member of genus Respirovirus of the family Paramyxoviridae.BPIV3 is a leading viral pathogen of Bovine respiratory disease syndrome,by which Bovine herpes virus I(BHV-I),Bovine respiratory syncytial virus(BRSV)or Bovine viral diarrhea virus infects(BVDV).BPIV3 causes significant economic losses to cattle industry every year worldwide.Serological surveys showed that BPIV3 has been widely distributed in China,therefore,it is imminent to establish a feasible and accurate method for diagnosis of disease associated with the BPIV3.In etiological diagnosis,in this study,one pair of specific primers and Taq Man-MGB probes were designed based on the sequences of BPIV3 published in Gen Bank to establish SYBR Green I Q-PCR and Taq Man-MGB Q-PCR methods,which could detect BPIV3 specifically.The specificity,sensitivity and repeatability of such two methods were evaluated.The standard curve in a range of 103~107 copies/μL showed a good linear relationship.In the specificity tests,both SYBR Green I Q-PCR and Taq Man-MGB Q-PCR methods were highly specific for detection of BPIV3 a and BPIV3 c,and neither of two methods was positive for bovine viral diarrhea virus and bovine herpes virus type I.In the sensitivity tests,the minimum detectable quantity of Taq Man Q-PCR was 1.0×101 copies/μL,in comparison,SYBR Green I Q-PCR was 1.0×103 copies/μL.In the repeatability tests,the Ct value variation coefficients of two methods were less than 1.0%.In serum diagnostics,in this study for development of an indirect ELISA,the nucleocapsid(NP)protein of BPIV3 was chosen,since it was relatively conservative in various BPIV3 strains.According to papers and software analysis of NP,the amino terminal of NP protein(aa 8~156)and the carboxyl terminal of NP protein(aa 368 ~507)were analyzed as the best epitope antigen regions.Two pairs of specific primers were designed to amplify the given target fragments.The prokaryotic expression vector p ET-28a-NP-N-C was constructed and expressed in E.coli BL21(DE3).The fusion protein NP-N-C was used as coating antigen by detection of BPIV3-positive and negative sera,which was developed for the detection of BPIV3 sera.The reaction conditions of indirect ELISA,including quantity of coating antigen and incubation time,dilution of the analyzed sera,selection of blocking buffer,dilution of secondary antibody and substrate incubation time were optimized.The ROC curve was constructed by SPSS analysis and cut-off value was determined.The specificity and sensitivity of the indirect ELISA were evaluated and its repeatability was verified.The optimal reactions were as follows: quantity of coating antigen was 4 μg/m L,incubated at 37℃for 1 h,followed by being incubated at 4℃ for 12 h.The dilution multiple of sera was 1:80.The concentration of blocking buffer was 5% skimmed milk and the reaction conditions of skimmed milk was incubated at 37℃for 1 h.The optimal reaction conditions of serum was incubated at 37℃for 1 h.The best dilution of secondary antibodies was 1:5000,the optimal reaction conditions of secondary antibodies was incubated at 37℃for 1 h.The incubation time of TMB substrate was 15 min.The cut-off value of OD450 reading was set as 0.267 and the resulting specificity of the indirect ELISA was 97.4% and the sensitivity was 95.3%.In the repeatability tests,the given sera were detected under optimal parameter,demonstrating that the coefficient of variations intra-assay and inter-assay was less than 10%.The Q-PCR assays established in this study represented a potential of detection of BPIV3 three genotypes and it provided a more rapid,stable and feasible method for the early diagnosis and quantitative analysis of BPIV3.The established indirect ELISA for detection of BPIV3 antibodies could be applied to serological epidemiological investigation and retrospect diagnosis for BPIV3.Q-PCRs and indirect ELISA developed in this study may provide the basic guide for the prevention and control of the disease due to BPIV3.