A SYBR Green I-based real-time polymerase chain reaction assay for detection and quantification of canine bufavirus

Canine bufavirus (CBuV) was first discovered in puppies in Italy in 2016, and subsequent studies have reported its possible relationship with acute enteritis. Currently, there is no specific and quantitative detection method for CBuV. This study examined the conserved NS1 gene and used a pair of specific primers to establish a direct SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) method for the detection and quantification of CBuV. In the sensitivity experiment, the detection limit of SYBR Green I-based real-time qPCR was 4.676 × 10¹ copies/μL and that of conventional PCR (cPCR) was 4.676 × 10³ copies/μL. Furthermore, the qPCR method did not detect other viruses in dogs, indicating good specificity. The intra-assay coefficient of variation was 0.07-0.55% and the inter-assay coefficient of variation was 0.03-0.11%, indicating good repeatability. In clinical sample testing, the detection rate of qPCR was 5.0% (6/120), higher than that of cPCR (2.5%, 3/120). In addition, the samples that tested CBuV-positive in this experiment were all from dogs with acute enteritis. In summary, the SYBR Green I-based qPCR method established in this study has good sensitivity, specificity, and reproducibility for clinical sample detection and can also assist in future research on CBuV.