Establishment and application of a real‑time fluorescent quantitative PCR assay for Bovine Rotavirus

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 °C 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 C_t 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.