Development of duplex crystal digital PCR (dPCR) assay for detection and differentiation of NDRV and MDRV

Muscovy duck reovirus (MDRV) and Novel duck reovirus (NDRV) are highly infectious diseases of waterfowl, causing significant harm to the global poultry industry. Early detection and diagnosis of NDRV and MDRV in clinical samples are crucial for effectively preventing and controlling these diseases. This study developed a duplex crystal digital PCR (dPCR) assay for the differential detection of NDRV and MDRV. Primer pairs and probes were designed specifically for the S3 genome of NDRV and the S2 genome of MDRV. To evaluate the method's performance, different reaction conditions were optimized, focusing on specificity, sensitivity, and reproducibility. The results showed that duplex crystal dPCR could accurately and differentially detect NDRV and MDRV, with a detection limit as low as 1 × 10⁻¹ copies/μl. It did not cross-react with other avian viruses, including duck Tembusu virus (DTMUV), H5 subtype avian influenza virus (H5 subtype AIV), H7 subtype AIV, H9 subtype AIV, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), Egg drop syndrome virus (EDSV), Contagious bursal disease virus (IBDV, B87 strain), and duck Plague virus (DEV). The method also demonstrated good reproducibility, with intra-assay and inter-assay coefficients of variation (CV) both less than 8 %. For clinical application, 299 samples from coastal areas of Guangxi, China were tested. Duplex crystal dPCR detected NDRV and MDRV positive rates of 8.027 % and 6.020 %, respectively, with a co-infection rate of 1.672 %. The kappa values between duplex crystal dPCR and duplex quantitative polymerase chain reaction (qPCR) were 0.977 for NDRV and 1 for MDRV, indicating strong agreement. These findings confirm that the established duplex crystal dPCR is a specific, sensitive, and accurate method for detecting and quantifying NDRV and MDRV. This is the first report of using duplex crystal dPCR for NDRV and MDRV detection.