Triplex fluorescence quantitative PCR detects duck hepatitis A virus type 3, Tembusu virus and novel reovirus

Mixed infections are a serious problem causing significant economic losses in the duck industry. Duck hepatitis A virus serotype 3 (DHAV-3), duck Tembusu virus (DTMUV) and novel duck reovirus (NDRV), belonging to the family Picornaviridae, Flaviviridae and Spinareoviridae, respectively, are important pathogens with similar symptoms in infected ducks. To detect and differentiate these viruses, this study developed a triplex real-time PCR assay targeting the VP1, E and σC genes of DHAV-3, DTMUV and NDRV, respectively. Validation tests and clinical applications followed the optimization of the triplex real-time PCR assay. The novel triplex assay showed high specificity with no cross-reactivity with eight other common duck viruses. The detection limits were 1 × 101 copies/μL for DTMUV, DHAV-3 and NDRV, with intra- and inter-assay variation coefficients (CV) below 4%. A total of 135 clinical samples were tested to evaluate the method's feasibility. The positive rates for DHAV-3, DTMUV and NDRV were 13.33%, 10.37% and 8.15%, respectively. Using virus isolation as a reference, the triplex qPCR assay showed 100% detection sensitivity for all three viruses, with specificity and coincidence values above 97%. In conclusion, the established triplex TaqMan PCR is highly specific, sensitive and reproducible, providing a practical approach for detecting and monitoring DHAV-3, DTMUV and NDRV in ducks.