Development and validation of a crystal digital PCR assay for duck Tembusu virus detection and vaccine monitoring.

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 (2.68×101 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.