Simultaneous detection of feline parvovirus and feline bocavirus using SYBR Green I-based duplex real-time polymerase chain reaction

Since both feline parvovirus (FPV) and feline bocavirus (FBoV) can cause diarrhea in cats, it is difficult to distinguish them clinically. This study aimed to develop a SYBR Green I-based duplex real-time polymerase chain reaction (PCR) assay for distinguishing FPV and FBoV-1 on the basis of the melting temperature of the PCR product. A total of 132 fecal samples from different domestic and feral cats were collected, and the results of SYBR Green I-based duplex real-time PCR assay were compared with those of the traditional PCR assay for a comprehensive evaluation. The melting temperatures were found to be 86 °C and 77.5 °C for FBoV-1 and FPV, respectively, and no specific melting peaks for other non-targeted feline viruses were observed. The data obtained from this assay had a good linear relationship; the detection limits of FPV and FBoV-1 were 2.907 × 101 copies/μL and 3.836 × 101 copies/μL, respectively. In addition, the experiment exhibited high reproducibility. The positive detection rates of the SYBR Green I-based duplex real-time PCR assay for FPV and FBoV-1 were 16.67% (22/132) and 6.82% (9/132), respectively, and the positive detection rate for co-infection with FPV and FBoV-1 was 3.03% (4/132). This result was much more sensitive than that of the traditional PCR method. Thus, the developed SYBR Green I-based assay is a sensitive, rapid, specific, and reliable method for the clinical diagnosis of FPV and FBoV-1 and can provide technical support for the simultaneous detection of co-infection with these viruses in the future.