Development and comparison of duplex crystal digital PCR and qPCR assays for the detection of Muscovy duck parvovirus and goose parvovirus.

Muscovy duck parvovirus (MDPV) and Goose parvovirus (GPV) are highly contagious waterfowl viruses causing significant harm to the poultry industry. This study designed primers and probes targeting the non-structural genomic region of MDPV and GPV, optimizing amplification for both quantitative polymerase chain reaction (qPCR) and duplex crystal digital polymerase chain reaction (dPCR). The resulting duplex crystal dPCR method showed strong specificity, detecting MDPV and GPV without cross-reactivity with other waterfowl viruses. Sensitivity tests revealed that the lower detection limit for qPCR was 11.5 copies/μL, while duplex crystal dPCR achieved a significantly lower detection limit of 0.3 copies/μL. This indicates that dPCR is approximately 38.3-fold more sensitive than qPCR in detecting MDPV and GPV The method demonstrated good repeatability with variation coefficients below 8.0%. In 52 clinical samples from Guangxi, the positive rates for MDPV and GPV were 15.38% and 23.08%, respectively, with one co-infection. This study confirms that duplex crystal dPCR is a specific, sensitive, and efficient technique for detecting MDPV and GPV, especially in the detection of samples containing low concentrations of viruses.