Abstract: Bovine kobuvirus (BKoV) is a potential diarrhea-causing pathogen in calves, and there is no real-time RT-PCR assay for detecting this virus until now. By designing primers targeted to 3D fragment of BKoV strains and optimizing reaction conditions and system, a TB Green real-time RT-PCR assay was successfully developed. The results demonstrated that the assay had a good linear relationship between 4.86×10⁸ and 4.86×10² copies·μL^-1 of virus concentration, the linear correlation coefficient was 0.999 5 and the amplification efficiency was 92.75%. This assay was only specific for detection of BKoV, no cross reaction was observed with other species of diarrhea-causing pathogens; the minimum detection limit was 4.86×10² copies·μL^-1; both the intra- and inter-group coefficients of variation were <2%. Comparing to other three reported RT-PCR assays, the assay had a significant high detection rate of BKoV for clinical feces samples from both diarrheal cows and yak. Total 131 yak diarrhea samples collected from Qinghai, Xizang and Sichuan region between May 2018 and May 2019, the BKoV detection rate was 31.30%. The real-time RT-PCR assay for detecting BKoV developed in this study has a good specificity and stability, which provide an effective method for detection and epidemiological investigation of BKoV.

Key words: bovine kobuvirus, real-time RT-PCR, yak, diarrhea