The Multiplex qPCR Assay Enables Simultaneous Detection, Differentiation, and Quantification of Feline Bocavirus-1,...

Feline bocaviruses (FBoVs) are increasingly recognized for their global distribution and potential importance in feline health. Three distinct species-FBoV-1, FBoV-2, and FBoV-3-have been identified; however, the lack of efficient molecular assays has limited the ability to clarify their species distribution, prevalence, and viral loads. In this study, we developed a multiplex quantitative PCR (qPCR) assay targeting the NS1 gene for the simultaneous detection, differentiation, and quantification of all three FBoV species. The assay demonstrated high analytical sensitivity, with limits of detection of 1.6 × 10³ copies/μL for FBoV-1 and 1.6 × 10² copies/μL for both FBoV-2 and FBoV-3. No cross-reactivity was observed with other feline or non-feline viruses, and the assay showed strong repeatability and reproducibility. Diagnostic evaluation using 135 archived feline oropharyngeal and fecal samples yielded a sensitivity of 95.2% and a specificity of 100% compared with previously established singleplex qPCR assays. Importantly, the multiplex format reduced time, cost, and labor while maintaining high diagnostic accuracy. This study presents the first multiplex qPCR assay capable of simultaneously detecting, differentiating, and quantifying all three FBoV species, providing a rapid, sensitive, and specific tool for routine diagnostics and large-scale epidemiological investigations.