The RT-RPA-CRISPR/Cas13a assay detected PRRSV-2 RNA at concentrations as low as 6 copies/μL, achieving a 28-fold increase in sensitivity compared to existing CRISPR/Cas13a-based methods.
The novel multiple-crRNA RT-RPA-CRISPR/Cas13a assay provides a highly sensitive, specific, and rapid visual detection method for PRRSV-2 that matches the accuracy of RT-qPCR.
Effect estimate: 28-fold increase in sensitivity
Absolute Event Rate: 6% vs 172%
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.
Guo et al. (2026) studied Porcine reproductive and respiratory syndrome virus (PRRSV) (n=12). RT-RPA-CRISPR/Cas13a assay vs. RT-qPCR and existing CRISPR/Cas13a methods was evaluated on Limit of detection (LOD) of PRRSV-2 RNA (28-fold increase in sensitivity). The RT-RPA-CRISPR/Cas13a assay detected PRRSV-2 RNA at concentrations as low as 6 copies/μL, achieving a 28-fold increase in sensitivity compared to existing CRISPR/Cas13a-based methods.