Development and validation of an LNA-based one-step multiplex RT-qPCR assay for differentiating Betaarterivirus europensis (PRRSV-1), Betaarterivirus americense (PRRSV-2), and the highly pathogenic L8 lineage of PRRSV-2.

LNA-based multiplex RT-qPCR enables rapid, accurate differentiation of PRRSV variants for enhanced surveillance.

This study presents a novel one-step multiplex RT-qPCR assay utilizing locked nucleic acid (LNA) technology for simultaneous detection and differentiation of three PRRSV variants: Betaarterivirus europensis (PRRSV-1), Betaarterivirus americense (PRRSV-2), and the highly pathogenic L8 lineage of PRRSV-2, with integrated RNA internal control. The assay demonstrated excellent analytical performance with R² values of 0.99 across all channels and low detection limits (13-26 copies/reaction at 95% probability). Validation included testing on spiked swine serum, naturally positive samples, and independent laboratory comparison against commercial standards. The assay proved highly robust, maintaining stability through temperature fluctuations, bench exposure, and freeze-thaw cycles while demonstrating minimal cross-reactivity and low intra/inter-assay variability. Qualitative agreement with comparator assays was 100%, and reagent stability persisted for 30 weeks. This multiplex platform addresses the critical diagnostic need for accurate PRRSV differentiation in surveillance and eradication programs, offering superior operational convenience compared to sequential testing approaches.

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