Abstract
Duck Tembusu virus (DTMUV) threatens the duck industry and public health due to its broad host range. This study established and validated a crystal digital PCR (cdPCR) assay for molecular detection of DTMUV, comparing it with quantitative real-time PCR (qPCR). A recombinant plasmid standard (pDTMUV, targeting the DTMUV E gene) served as a reference. Optimized at 58°C annealing temperature with optimal primer/probe concentrations, cdPCR showed high specificity (only DTMUV positive) and precision (low intra-/inter-assay CVs), with a limit of detection (LOD) of 0.19 copies/μL-100-fold more sensitive than qPCR (2.68×10(1) copies/μL). In 326 Guangxi clinical samples, cdPCR detected a higher DTMUV positive rate (7.36% vs. qPCR's 6.75%) with high consistency (Kappa=0.955) and accurately identified low-viral-load samples. For vaccine monitoring, cdPCR tracked DTMUV in immunized chicks' plasma at 24, 72, and 168 h post-vaccination (revealing viral dynamics) and detected DTMUV in tissues of chicks euthanized 30 min post-injection (abundant at the injection site, with spread to blood/organs). This cdPCR assay is a reliable molecular detection tool for DTMUV clinical diagnosis and vaccine monitoring, with superior sensitivity for low-viral-load samples.