Objective  A rapid, accurate qRT-PCR method for detecting bovine kobuvirus (BKoV) was established and clinically tested for application.

  Method  A pair of specific primers and a probe were designed and synthesized according to the 3D gene sequence of BKoV published on GenBank for the establishment of a TaqMan fluorescence qRT-PCR assay. The reaction system of the analytic was optimized prior to verification on clinical specimens.

  Result  The optimal upstream and downstream primers were 300 nmol·L⁻¹ in concentration, and the applied probe 400 nmol·L⁻¹. A linearity with a correlation coefficient of R²= 0.999 was achieved in the range of 1×10¹−1×10⁸ copies·μL⁻¹. The amplification efficiency reached 103%. The assay demonstrated a high specificity among the pathogens related to diarrhea in calves, a high sensitivity with the minimum detection limit of 1×10¹ copies·μL⁻¹ on BKoV plasmid standard, in comparison to that of 1×10² copies·μL⁻¹ by conventional RT-PCR, and a high repeatability with a coefficient of variation of less than 3% within and between groups. In 37 bovine fecal samples collected from different pastures in Inner Mongolia from March to May 2021, the assay showed a positive detection rate of 24.3%. The calculated viral loads using the standard curve had an average on the feces from diarrheal animals at 3.7×10⁵ copies·μL⁻¹ and at 8.65×10³ copies·μL⁻¹ on the specimens from healthy calves.

  Conclusion  The newly developed TaqMan fluorescent qRT-PCR assay was specific, repeatable, and sensitive in detecting BKoV. It could be applied for clinic diagnosis and epidemiological investigation on the disease.