Development and validation of an LNA-based multiplex RT-qPCR assay for differentiating Betaarterivirus europensis...

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly variable arterivirus that causes major economic losses in swine production and requires reliable molecular diagnostics for surveillance and eradication programs. We developed and validated a one-step multiplex RT-qPCR assay for the simultaneous detection and differentiation of Betaarterivirus europensis (PRRSV-1), Betaarterivirus americense (PRRSV-2), and the highly pathogenic L8 lineage of PRRSV-2, together with an RNA internal control in a single reaction. Short LNA-modified probes were designed to target conserved yet discriminatory sequence motifs, improving specificity and supporting multiplex detection. Analytical performance was evaluated using spiked swine serum, naturally positive samples, and independent laboratory testing against a commercial comparator. The assay showed excellent linearity across all channels (R² = 0.99) and low detection limits of 13-26 copies per reaction at 95% detection probability. Robustness testing, including 2 h bench exposure and a 3°C thermocycler shift, produced negligible Cq changes, while a 1,000-fold competitor challenge indicated minimal cross-reactivity. Intra- and inter-assay variability were low, qualitative agreement with the comparator was 100%, and reagents remained stable for 30 weeks and after up to 10 freeze-thaw cycles. This LNA-based multiplex RT-qPCR assay provides a sensitive, specific, and operationally convenient tool for PRRSV surveillance and control programs.