A duplex SYBR Green I-based real-time PCR assay was established for the simultaneous detection of canine kobuvirus (CaKoV) and canine astrovirus (CaAstV). This assay can easily distinguish the two viruses according to their different melting temperatures (Tm) of 80 °C for CaKoV and 86.5 °C for CaAstV; other canine enteroviruses used as controls showed no specific melting peaks. The detection limit of this assay was determined to be 10¹ copies/μL for both viruses. This method exhibited high repeatability and reproducibility, with a coefficient of variation less than 1.5 %. A total of 48 fecal samples were collected for clinical testing by real-time PCR and confirmed by sequencing. Real-time PCR assay showed a 10.4 % CaKoV-positive rate and a 4.2 % CaAstV-positive rate, and the positive rate of co-infection of the two viruses was 2.1 %, which was consistent with the sequencing results. This assay has many advantages over conventional PCR: it is rapid, sensitive, specific, and reliable for detecting these two viruses in one sample, and it can be used as a tool to detect CaKoV and CaAstV infection or co-infection in clinical settings.

建立了基于双链 SYBR Green I 的实时 PCR 检测，用于同时检测犬科布病毒 (CaKoV) 和犬星状病毒 (CaAstV)。该测定可以根据两种病毒的不同熔解温度 (Tm) 轻松区分 CaKoV 为 80 °C，CaAstV 为 86.5 °C；用作对照的其他犬肠道病毒没有显示出特定的熔解峰。该测定的检测限确定为 10 ¹两种病毒的拷贝/μL。该方法具有高重复性和再现性，变异系数小于 1.5%。共收集了 48 份粪便样本，通过实时 PCR 进行临床检测，并通过测序确认。Real-time PCR检测显示CaKoV阳性率为10.4%，CaAstV阳性率为4.2%，两种病毒共感染阳性率为2.1%，与测序结果一致。该检测方法与传统 PCR 相比具有许多优势：它可以快速、灵敏、特异且可靠地检测一个样本中的这两种病毒，并且可以用作检测临床环境中 CaKoV 和 CaAstV 感染或合并感染的工具。