Development of a TaqMan-Based Duplex Real-Time Quantitative RT-PCR for Detection and Differentiation of Muscovy Duck Reovirus and Novel Duck Reovirus

Muscovy duck reovirus (MDRV) and novel duck reovirus (NDRV) are major pathogens in duck breeding, leading to substantial economic losses in the waterfowl industry. This study aimed to develop a precise detection and differentiation method for both viruses simultaneously. Specific primers and probes targeting the S3 gene were designed, and a duplex TaqMan-based real-time RT-PCR assay was established following optimization of reaction conditions. The assay demonstrated high amplification efficiency (100.1-106.7%), strong linear correlation (R² > 0.999), and low limits of detection (13-25 copies/µL). Intra- and inter-assay coefficients of variation were below 1.5%, confirming excellent repeatability and stability. Applied to 122 clinical duck tissue samples, the assay detected MDRV in 29.5% (36/122) and NDRV in 39.3% (48/122) of samples, with results fully validated by singleplex RT-PCR assays. Our study provides a reliable, specific, and reproducible tool for surveillance and epidemiological studies of MDRV and NDRV.