牛轮状病毒实时荧光定量PCR检测方法的建立及应用OA

Specific primers were designed based on the conserved sequences of the VP7 gene of Bovine rotavirus(BRV)retrieved from the NCBI database.By optimizing the reaction system and amplification conditions,a SYBR Green Ⅰ RT-qPCR assay for BRV detection was established.Experimental results showed that the optimal annealing temperature was determined as 52 ℃ and the optimal final concentration of the primers was 0.8 μmol/L.A good linear relationship(R²=0.998 9)was observed between the template concentration and the Ct value within the range of 3.16×10³～3.16×107 copies/μL,with the minimum detection limit of 3.16×10¹ copies/μL,which represented a 100 fold increase in sensitivity compared with the conventional PCR method.Specificity and repeatability verification tests indicated that the established assay produced specific amplification bands only for BRV,and no non-specific amplification signals were detected for other common pathogens causing calf diarrhea.The coefficients of variation(CVs)of both intra-assay and inter-assay were less than 1%.A total of 60 fecal samples suspected of BRV infection were detected,and the positive detection rate was 21.7%,which was significantly higher than that of the conventional PCR method(13.3%).In conclusion,the established RT-qPCR assay exhibited high specificity,sensitivity and repeatability,and could be applied for the rapid clinical detection of BRV and the development of diagnostic kits.