A SYBR Green I-based real-time polymerase chain reaction assay for detection and quantification of canine bufavirus. (October 2021)

• Record Type: Journal Article
• Authors: Wang, Yong; Sun, Jianfei; Guo, Xu; Li, Wei; Zhang, Da; Liu, Guangqing; Zhou, Tianhong; Li, Yongdong
• Is Part Of: Molecular and cellular probes. Volume 59(2021)
• Publication Date: 2021-10
• Subjects: Canine bufavirus -- SYBR Green I -- Real-time polymerase chain reaction -- Quantitative analysis; Molecular probes -- Diagnostic use -- Periodicals; Pathology, Cellular -- Technique -- Periodicals; Cell Biology -- Periodicals; Molecular Biology -- Periodicals; Sondes moléculaires -- Utilisation diagnostique -- Périodiques; Cytopathologie -- Technique -- Périodiques; 572
• DOI: 10.1016/j.mcp.2021.101762
• Languages: English
• ISSNs: 0890-8508

Abstract

Abstract: 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 × 10 1 copies/μL and that of conventional PCR (cPCR) was 4.676 × 10 3 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. Highlights: The study established a SYBR Green I-based real-time RT-PCR assay for Canine Bufavirus detection. The assay is highly sensitive, specific, and reproducible. It provides a tool for clinical diagnostics of Canine Bufavirus infection.