Development and application of a multiplex qPCR assay for the detection of duck circovirus, duck Tembusu virus, Muscovy duck reovirus, and new duck reovirus.

A multiplex qPCR assay was developed to simultaneously detect duck circovirus (DuCV), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and novel duck reovirus (NDRV), but it did not amplify other viruses, including duck virus enteritis (DVE), infectious bursal disease virus (IBDV), avian reovirus (ARV), H5 avian influenza virus (H5 AIV), H7 avian influenza virus (H7 AIV), H9 avian influenza virus (H9 AIV), Newcastle disease virus (NDV), and Muscovy duck parvovirus (MDPV), and the detection limit for DuCV, DTMUV, MDRV, and NDRV was 1.51 × 101 copies/μL. The intra- and interassay coefficients of variation were less than 1.54% in the repeatability test with standard plasmid concentrations of 1.51 × 107, 1.51 × 105, and 1.51 × 103 copies/μL. The developed multiple qPCR assay was used to examine 404 clinical samples to verify its practicability. The positivity rates for DuCV, DTMUV, MDRV, and NDRV were 26.0%, 9.9%, 4.0%, and 4.7%, respectively, and the mixed infection rates for DuCV + DTMUV, DuCV + MDRV, DuCV + NDRV, MDRV + NDRV, DTMUV + MDRV, and DTMUV + NDRV were 2.7%, 1.2%, 1.2%, 1.0%, 0.5%, and 0.7%, respectively.