Multiplex on-site detection of porcine diarrheal viruses via a quantum dot microspheres-based RT-RAA-CRISPR lateral flow platform

Porcine diarrhea caused by viral pathogens such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine group A rotavirus (PoRVA) results in substantial economic losses in the global swine industry. Rapid, accurate, and field-deployable detection is critical for early diagnosis and timely outbreak intervention, particularly in resource-limited settings. In this work, we developed an integrated point-of-care diagnostic platform combining reverse transcription recombinase-aided amplification (RT-RAA), CRISPR/Cas12a-mediated nucleic acid detection, and quantum dot microspheres (QDMs)-based fluorescent parallel lateral flow assays for the multiplex detection of PEDV, PDCoV, and PoRVA. Moreover, a smartphone-compatible portable fluorescence reader was incorporated to enable quantitative analysis in field applications. The developed system enables rapid detection within one hour, with a limit of detection (LOD) as low as 1 copy/μL. It also demonstrates excellent specificity without cross-reactivity to other tested viral pathogens, including ASFV, CSFV, TGEV, PRRSV, SADS-CoV, and BRV, and maintains stable performance after storage at 37℃ for one month. Validation with simulated, mixed-infection, and clinical samples demonstrated 100 % concordance with the proposed system and conventional RT-qPCR assays. This novel platform offers a rapid, ultrasensitive, stable, and user-friendly solution for on-site monitoring and early diagnosis of porcine viral diarrhea, providing an effective tool for disease surveillance and control in swine breeding. Copyright © 2026. Published by Elsevier B.V.