Development and clinical validation of a multiplex visual POCT platform based on RT-RAA-CRISPR/Cas12a for porcine diarrheal viruses

Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine sapelovirus (PSV) are major causative agents of severe diarrhea in swine and frequently occur as co-infections, leading to significant financial losses. To address the urgent demand for rapid, on-site, and multiplex detection, we developed a multiplex RT-RAA-CRISPR/Cas12a assay for visual point-of-care (POCT) diagnosis. The assay exhibited high specificity for the three target viruses, with no cross-reactivity against non-target swine pathogens. Under optimized conditions, the system achieved single-copy-level sensitivity, with limits of detection (LOD) of 5.2, 5.9, and 0.57 copies/μL for PEDV, PoRV, and PSV, respectively, and demonstrated excellent repeatability (CV < 10%). Validation with 20 clinical fecal swabs samples and a portable detection toolbox revealed 95%-100% concordance with standard RT-qPCR and RT-PCR methods. The entire "sample-to-answer" workflow was completed within 55 minutes. This platform provides a powerful and user-friendly tool for POCT of mixed viral infections in swine herds.