Development of a Triplex TaqMan Real-Time PCR Assay for Simultaneous Detection of Duck Hepatitis B Virus, Duck Adenovirus Type 3, and Streptococcus gallolyticus subsp. pasteurianus.

Duck hepatitis B virus (DHBV), Duck adenovirus 3 (DAdV-3) and Streptococcus gallolyticus subsp. pasteurianus (SGSP) are three distinct pathogens associated with infectious diseases in waterfowl. Clinical differentiation among these agents is often difficult due to overlapping signs, underscoring the need for accurate, rapid, and simultaneous diagnostic tools to support early detection and effective disease surveillance. In this study, we developed and validated a triplex TaqMan real-time quantitative PCR (qPCR) assay for the simultaneous detection and quantification of DHBV, DAdV-3, and SGSP. The assay indicated high analytical sensitivity, achieving detection limits of 10 copies/µL for each target, with all standard curves showing excellent linearity (R2 = 0.999). The evaluation of specificity showed that the triplex qPCR assay had high specificity for the three target pathogens, as it did not yield any cross-reactive signals with other pathogens examined. Intra-assay and inter-assay coefficients of variation for recombinant plasmid standards were consistently <2%, indicating high reproducibility. Meanwhile, the assay was employed to investigate the epidemiology of the three pathogens in ducks. The positive rates for DHBV, DAdV-3 and SGSP were 73.95% (159/215), 11.16% (24/215) and 37.21% (80/215), respectively. The co-infection rates were 5.58% (12/215) for DHBV and DAdV-3, 27.91% (60/215) for DHBV and SGSP, 2.33% (5/215) for DAdV-3 and SGSP, and 2.33% (5/215) for all three pathogens. In summary, this triplex qPCR assay displays excellent specificity, sensitivity and repeatability, which may serve as a reliable testing tool for routine clinical diagnosis, epidemiological monitoring and co-infection screening of the three target pathogens in locally sampled duck populations.