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• The first development a SYBR Green I-based real-time PCR assay to detect canine kobuvirus. • Canine kobuvirus (CaKoV) was detected at a rate of 8.7 % in the Anhui province. • This method has implications for the prevention and control of CaKoV-infect in dogs. Canine kobuvirus (CaKoV) is a causative agent of gastroenteritis in dogs. Rapid detection of CaKoV is important for preventing and controlling this condition. In this study, an SYBR Green I-based quantitative real-time PCR assay was established for CaKoV detection. Specific primers targeting a highly conserved region of the CaKoV 3D gene were developed. After optimization, the method detected a minimum of 1 × 101 copies/μL with high specificity, stability, and repeatability. Moreover, the entire process only required approximately 1.5 h for completion. Our results were supported by those obtained for clinical samples, in which our developed method was successfully applied. The newly established real-time PCR is a rapid, sensitive, specific, and repeatable method for the quantitative detection of CaKoV and can, therefore, be used in epidemiological studies. [ABSTRACT FROM AUTHOR]