Development of a Quadruplex RT-qPCR Assay for Rapid Detection and Differentiation of PRRSV-2 and Its Predominant Genetic Sublineages in China

The assay demonstrated high sensitivity (LOD of 3 copies/μL), high specificity, and good repeatability (coefficient of variation < 1.5%). Field application using 938 samples from Guangxi A and B farms revealed NADC30-like PRRSV wild-type strains at positivity rates of 13.44% and 3.53%, respectively. Positive samples selected for sequencing were further confirmed using ORF5-based phylogenetic analysis and NSP2 deletion pattern comparison, which aligned with RT-qPCR detection results. Field application primarily detected NADC30-like PRRSV, while further validation is still needed for HP-like and NADC34-like strains. The developed quadruplex RT-qPCR assay enables rapid and simultaneous detection of PRRSV-2 and differentiation of three major lineages, providing a sensitive, specific, and reliable tool for distinguishing vaccine-derived from circulating strains and supporting targeted disease surveillance and control in swine farms.