An advanced rapid-visual CRISPR assay for detecting porcine reproductive and respiratory syndrome virus

The porcine reproductive and respiratory syndrome virus (PRRSV) remains a significant threat to the global swine industry, underscoring the urgent need for innovative diagnostic methods to detect and manage outbreaks effectively. We developed a novel CRISPR-based fluorescence assay for the highly sensitive detection of PRRSV-2. By combining reverse transcription-recombinase polymerase amplification (RT-RPA) with multiple-crRNA CRISPR/Cas13a system and single-stranded RNA-fluorescently quenched reporters (RQ-5U), our assay achieved a significant 28-fold increase in sensitivity compared to existed CRISPR/Cas13a-based PRRSV-2 detection methods. This multiple crRNA strategy allows detecting as low as 6 copies/µL of PRRSV-2 RNA, significantly improving the detection limit. Moreover, our method's accuracy in detecting simulated PRRSV-2 clinical samples matches that of quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate that this visual, sensitive, and specific nucleic acid detection method holds great promise for enhancing the diagnosis and management of PRRS in the swine industry.