Wang Yong, Sun Jianfei, Guo Xu, Li Wei, Zhang Da, Liu Guangqing, Zhou Tianhong, Li Yongdong
Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, PR China.
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, PR China.
Mol Cell Probes. 2021 Oct;59:101762. doi: 10.1016/j.mcp.2021.101762. Epub 2021 Sep 2.
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 copies/μL and that of conventional PCR (cPCR) was 4.676 × 10 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.
犬杯状病毒（CBuV）于2016年在意大利的幼犬中首次被发现，随后的研究报告了其与急性肠炎可能存在的关系。目前，尚无针对CBuV的特异性定量检测方法。本研究检测了保守的NS1基因，并使用一对特异性引物建立了基于SYBR Green I的直接实时定量聚合酶链反应（qPCR）方法，用于检测和定量CBuV。在灵敏度实验中，基于SYBR Green I的实时qPCR的检测限为4.676×10拷贝/μL，传统PCR（cPCR）的检测限为4.676×10拷贝/μL。此外，qPCR方法未检测到犬的其他病毒，表明特异性良好。批内变异系数为0.07 - 0.55%，批间变异系数为0.03 - 0.11%，表明重复性良好。在临床样本检测中，qPCR的检出率为5.0%（6/120），高于cPCR（2.5%，3/120）。此外，本实验中检测为CBuV阳性的样本均来自患有急性肠炎的犬。综上所述，本研究建立的基于SYBR Green I的qPCR方法在临床样本检测中具有良好的灵敏度、特异性和可重复性，也可为今后CBuV的研究提供帮助。
