Simultaneous detection of novel goose parvovirus and novel duck reovirus by SYBR Green I-based duplex real-time quantitative polymerase chain reaction

Co-infection with novel goose parvovirus (NGPV) and novel duck reovirus (NDRV) is common, significantly impeding duck growth and resulting in considerable economic losses within the duck farming industry. To facilitate rapid and accurate diagnosis and differentiation of these two viruses, this study developed a SYBR Green I-based duplex real-time quantitative polymerase chain reaction (qPCR) assay. This assay enabled the simultaneous detection of NGPV and NDRV by exploiting their distinct melting temperatures (Tm): 78.5 ± 0.50 °C for NGPV and 84.5 ± 0.50 °C for NDRV. No amplification was observed for other prevalent non-target duck viruses. The intra- and inter-assay coefficients of variation were less than 1.75%. The assay showed good performance with the same detection limit of 10² copies/μL for both NGPV and NDRV. The results of the clinical testing indicated that 45.3% (34/75) of the samples tested positive for NGPV, while 38.7% (29/75) were positive for NDRV. Notably, 13.3% (10/75) exhibited co-infection. These results revealed that the sensitivity of the developed method exceed that of conventional polymerase chain reaction (PCR). The developed method for the identifying of NGPV and NDRV shows good specificity, sensitivity, and repeatability, rendering it an effective tool for the simultaneous detection of co-infection with NGPV and NDRV.