Development And Application Of A SYBR Green ⅠReal-time PCR Assay For Detection Of Bovine Coronavirus

Bovine Coronavirus, BCoV belongs to the coronavirus family. As a kind of2asubgroup, Conronavirus is a major pathogen causing neonatal calf diarrhea and adultcattle dysentery and respiratory infection. Epidemiological surveys show that BCoVis prevalent in cattle, mainly transmitted through the digestive tract and respiratorytract. Infected cattle experience long-term BCoV and sustained detoxification over aperiod of time that could easily lead to large-scale cattle infection. Thus, timelydetection of infected cattle and cattle farm environmental monitoring are particularlyimportant. There is no specific drug for the treatment of BCoV infection at home andabroad. Countries with developed dairy industry apply inactivated vaccines orattenuated vaccine to prevent the disease, but there is no related commercial vaccinein China. Therefore, it is urgent to establish rapid and sensitive detection methods ofBCoV, and necessary to investigate BCoV prevalence in the dairy farms of some areasin China so that the cattle infected BCoV can be early detected and epidemiologicaldata be mastered. In this way, we can take timely and effective correspondingmeasures of prevention and control, in order to decrease the economic losses causedby the virus to the cattle industry.According to BCoV N gene sequences included in GenBank, this study designsand synthesizes a pair of specific primers of amplified N gene’s partial fragments. Theprocess is: take BCoV’s cDNA as a template for PCR amplification. After purifiedPCR products are recovered, send to BGI (Beijing Genomics Institute) for genesequencing. Connect correct-sequencing N gene fragments to pEASY-T3vector.Confirmed by enzyme digestion and sequencing validation, recombinant plasmid pEASY-T3-N is obtained. Take the obtained recombinant plasmid pEASY-T3-N aspositive standard substance to conduct a series of dilutions. Then select six standardsubstances with gradient dilutions as a template for fluorescent quantitative PCRreaction to establish the standard curve and the regression equation. After then useconventional PCR and real-time fluorescent quantitative PCR method to make aaetiology detection of212clinical samples collected from5large-scale dairy farms byour laboratory in2013-2014. Finally, a pair of specific primers used for theamplification of N gene’s full length are designed and synthesized. Send the productafter PCR amplification for sequencing identification. And at last, make aphylogenetic analysis between the N gene’s full length sequencing measured bySNAatar biology software and the sequencing in GenBank.Sequencing shows that the fragment size of100bp by clone is right andrecombinant plasmid pEASY-T3-N positive standard substance is successfullyobtained. In the established detection method of BCoV GreenⅠreal-time fluorescentquantitative PCR, template’s lowest detection limit in a reaction system is7.8copies/μL, which is more sensitive than conventional RT-PCR; melting curve only appears aspecific single peak, without obvious primer dimers or nonspecific amplificationpeaks, showing that the method has good specificity. Concurrently it has no crossreaction with BRV, BVDV, IBRV, BEV, BEFV and BPIV-3; both intra-assay andinter-assay coefficients of variation were less than1.5repeat%. Therefore, themethod is with high sensitivity and specificity and good repeatability, providingtechnical assistance for clinical diagnosis and epidemiology survey of BCoV.The PCR detection of clinical samples shows that: in107nasal swab samples,BCoV infection positive rate is5.61%, and BCoV’s mixed positive infection rateswith BVDV, IBRV and BPIV3are4.60%,4.70%and5.60%; in105fecal samples,BCoV infection positive rate is33.33%; and BCoV’s mixed positive infection rateswith BVDV, IBRV, BPIV are18.10%,5.71%,29.52%. The above survey shows thatBCoV is widespread in cattle farms, and has a serious mixed infection with otherpathogens, bringing significant risks to the cattle disease prevention. Throughamplification of BCoV N gene’s full length sequence in positive samples, plus sequencing analysis, it discovers that the full length sequence between the N geneafter measurement and the N gene included in GenBank has little difference.