The major cause of canine gastroenteritis is viral infection, especially in dogs under one year of age. The main clinical symptom of canine gastroenteritis is diarrhea, and the common viruses that cause diarrhea in dogs are CPV, CDV and CCV[19–21]. In addition, there are two new viruses, CaKoV and CaCV, which can also cause diarrhea in dogs[8, 13, 22]. CaKoV and CaCV infection is a serious threat to the health of dogs, the mortality rate is as high as 50–100%, which has caused huge economic losses to the canine industry. CaKoV has been detected in cattle, sheep, pigs, and dogs in different countries[24–27]. Since circovirus was first identified in the United States in 2012, it has been reported in Italy, Germany, China, Thailand and other countries[15, 22, 28–30]. Because the clinical symptoms of the two viruses are very similar, it is difficult to differentiate and diagnose the two viruses[17, 23]. Therefore, a test that simultaneously detects and identifies these two viruses is required.
Viruses are detected in a variety of ways, such as loop-mediated isothermal amplification, enzyme-linked immunosorbent assays, and indirect immunofluorescence assays[31–34]. Compared to these traditional detection methods, real-time fluorescence quantitative PCR has great advantages in terms of sensitivity and specificity[33, 35], and detects virus even at lower concentrations. In addition, compared with TaqMan-based real-time PCR, the SYBR Green I-based real-time PCR method is cheaper and easier. Compared to a single assay, a dual assay was designed with the same sensitivity, but with the ability to detect multiple viruses. Therefore, a duplex SYBR Green I-based real-time PCR was developed that simultaneously detected CaKoV and CaCV.
In this study, a duplex SYBR Green I-based real-time PCR assay was successfully established. Two specific primers were designed based on the conserved regions, 3D for CaKoV and Rep for CaCV. The method distinguished the two viruses by their different Tm values, which were 86°C for CaKoV and 78°C for CaCV. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/µL and 3.841 × 101 copies/µL, respectively, demonstrating the high sensitivity of the method. Moreover, when this method was used to detect other viruses, such as CaAstV, CDV, CCV, and CPV, there was no obvious melting curve, indicating that the assay had good specificity. The intra -and inter-assay CV values were low, demonstrating that the experiment could be repeated with a high degree of reproducibility. Furthermore, the duplex SYBR Green I-based real-time PCR and conventional PCR assays were used to detect viruses within clinical samples. The positive infection rate of the former was significantly higher than that of the latter, indicating that the duplex SYBR Green I-based real-time PCR assay was more suitable for the detection of these two viruses.