Establishment of an LNA-TaqMan fluorescent quantitative PCR assay for differential diagnosis of virulent and attenuated strains of short beak and dwarfism syndrome virus.

To enable differential detection of virulent and attenuated strains of duck short beak and dwarfism syndrome virus (SBDSV), we developed a fluorescent quantitative PCR (qPCR) assay based on locked nucleic acid (LNA)-TaqMan probes. This assay utilized a pair of universal primers and two strain-specific LNA-TaqMan probes (labelled with FAM and VIC, respectively) targeting the VP1 gene of SBDSV. Validation results showed that the assay specifically detected SBDSV virulent and attenuated strains without cross-reacting with other common waterfowl viruses. It exhibited high sensitivity, with a minimum detection limit of 6.0 × 10⁰ copies/μl, and excellent reproducibility, with intra- and inter-assay coefficients of variation (CVs) all < 3%. Additionally, viral titre estimates from this qPCR assay showed no significant difference from those obtained via the gold-standard 50% tissue culture infectious dose (TCID₅₀) assay (P > 0.05). In conclusion, this LNA-TaqMan qPCR assay provides a specific, sensitive, and reliable tool for the differential diagnosis of SBDSV virulent and attenuated strains in clinical practice, serving as strong technical support for the prevention, control, and eradication of SBDSV.