Duplex SYBR Green I-based real-time PCR assay for the rapid detection of canine kobuvirus and canine astrovirus

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.