Abstract
The global swine industry suffers substantial economic losses from Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which causes reproductive and respiratory disorders. With NADC30- and NADC34-like strains now predominant, differentiation is complicated by their similar biology, despite characteristic Nsp2 gene deletions of 131 aa and 100 aa, respectively, highlighting a need for improved diagnostics. Moving fundamentally beyond conventional broad-spectrum detection, this assay innovatively targets the characteristic Nsp2 deletion signatures. A diagnostic platform integrating CRISPR-Cas13a with recombinase-aided amplification (RT-RAA) was constructed. Utilising aligned Nsp2 sequences, conserved crRNAs, and RT-RAA primers, NADC30- and NADC34-like strains were evaluated for their assay time, specificity, sensitivity, and consistency with RT-qPCR. The assay achieved rapid, specific detection with no cross-reactivity to common swine pathogens, and highly sensitive detection limits of 1.7 × 10¹ and 2.3 × 10¹ copies/µL for NADC30- and NADC34-like strains, respectively. Clinical validation using 30 samples demonstrated complete concordance with RT-qPCR, and the integration of T7 transcription enabled the single-tube differentiation of both strains. In summary, this one-tube, two-step fluorescent system presents a robust alternative for PRRSV diagnostics, combining specificity, sensitivity, and ease of use. It is well-suited for rapid field identification, molecular typing, and surveillance, thereby aiding in the implementation of targeted control measures.