Abstract
Novel duck reovirus (NDRV), duck hepatitis A type 1 virus (DHAV-1), duck Tembusu virus (DTMUV), and duck hepatitis A type 3 virus (DHAV-3) caused substantial economic losses within the duck industry. In our research, we aimed to develop a TapMan probe-based multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR) method to enable the early detection of these four viruses in suspicious duck populations. The detection limits for DTMUV, NDRV, DHAV-1, and DHAV-3 were 8.08 × 10¹, 1.24 × 10², 6.03 × 10¹, and 1.88 × 10² copies/μL, respectively. Additionally, the DTMUV, NDRV, DHAV-1, and DHAV-3 were successfully detected. No positive signals were detected for duck enteritis virus (DEV), muscovy duck parvovirus (MDPV), goose parvovirus (GPV), avian influenza virus (AIV), and newcastle disease virus (NDV). The correlation coefficient is greater than 0.99, and the amplification efficiency is 80-100 %. In the reproducibility tests conducted with standard plasmid concentrations at 10(7), 10(5), and 10(3) copies/μL, both the intra-assay and inter-assay coefficients of variation were below 10 %. The multiplex RT-qPCR method was used to detect 215 clinical samples. The positivity rates for NDRV, DHAV-3, DHAV-1, and DTMUV were 2.32 %, 6.27 %, 5.58 %, and 19.30 %. The positivity rates for DHAV-3 and DTMUV co-infection were 5.58 % for DHAV-3, DTMUV, and NDRV co-infection were 0.05 %.