A single-tube dual-gene RAA-PfAgo assay for simultaneous detection and identification of PEDV field and vaccine strains.

Porcine epidemic diarrhea virus (PEDV) poses a significant threat to the swine industry, and distinguishing circulating field strains from vaccine strains under field conditions remains challenging. In this study, we established a one-tube visual detection platform based on recombinase‑aided amplification (RAA) and PfAgo nuclease for simultaneous detection of the highly conserved N gene and the virulence‑associated ORF3 gene of PEDV. The assay completes within 60 min, with a sensitivity of 100 cDNA copies/μL. Specificity tests showed no cross‑reactivity with other porcine viruses tested. Using FAM‑ and ROX‑labeled probes, the N gene produces green fluorescence, ORF3 generated red fluorescence, and dual positivity appears yellow, which all visible to the naked eye under blue light. In validation with 36 clinical samples, 12 were identified as PEDV‑positive, including 3 field strains. Using RT‑PCR sequencing as the reference, the method achieved 100% specificity. Overall, this RAA‑PfAgo approach integrates rapidity, high sensitivity, specificity, and visualization without complex instrumentation, offering a powerful tool for on‑site clinical diagnosis, strain differentiation, and epidemiological monitoring in pig farms.