Abstract
Waterfowl parvoviruses (WPVs), including classical goose parvovirus (cGPV), Muscovy duck parvovirus (MDPV), Muscovy duck-origin goose parvovirus (MDGPV), and short beak and dwarfism syndrome virus (SBDSV), are significant pathogens that affect waterfowl flocks worldwide. Due to their high genetic similarity and frequent coinfections, rapid and accurate differentiation of these viruses remains challenging. In this study, we developed a multiplex TaqMan-minor groove binder (MGB) real-time PCR assay for the simultaneous detection and differentiation of cGPV, MDPV, MDGPV, and SBDSV. Specific primers and TaqMan-MGB probes were designed based on sequence alignments of the VP gene. This assay exhibited high specificity, with no cross-reactivity to other main waterfowl viruses. The detection limits of this assay were 102 copies/μL for cGPV, 101 copies/μL for MDPV, 102 copies/μL for MDGPV, and 10³ copies/μL for SBDSV, respectively. The standard curves exhibited strong linearity (R2≥0.995) and high amplification efficiency (89%-108%), with intra- and interassay coefficients of variation below 2.0%, indicating high repeatability and stability. Clinical testing of 337 clinical samples suspected of WPV infection demonstrated that the developed assay outperformed conventional PCR, achieving higher overall detection rates (58% vs 54%) and enhanced identification of coinfections. Epidemiological analysis revealed MDGPV as the predominant circulating strain in Muscovy ducks, with 27 samples identified as coinfected with both MDGPV and MDPV, while SBDSV showed higher prevalence in mule ducks and Pekin ducks. Notably, MDGPV was detected for the first time in goslings. These findings provide clear evidence of ongoing host restriction and potential cross-species transmission of WPVs among duck flocks. In conclusion, the multiplex TaqMan-MGB quantitative PCR (qPCR) assay developed in this study provides a rapid, sensitive, and reliable tool for the simultaneous detection and differentiation of cGPV, MDPV, MDGPV, and SBDSV. Its application is expected to enhance disease surveillance, facilitate outbreak control, and contribute to more effective control of waterfowl parvoviral diseases.