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

Porcine epidemic diarrhea virus (PEDV), porcine rotavirus A (PoRVA), 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 reverse transcription recombinase-aided amplification (RT-RAA)-CRISPR/Cas12a assay for visual point-of-care testing (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, PoRVA, and PSV, respectively, and demonstrated excellent repeatability (coefficient of variation (CV) < 10%). Validation with 20 clinical fecal swabs samples and a portable detection toolbox revealed 95%-100% concordance with standard reverse transcription quantitative polymerase chain reaction (RT-qPCR) and reverse transcription polymerase chain reaction (RT-PCR) methods. The entire "sample-to-answer" workflow was completed within 55 min. This platform provides a powerful and user-friendly tool for POCT of mixed viral infections in swine herds.