Multienzyme isothermal rapid amplification (MIRA)-LFD assay for rapid detection of PEDV and PoRVA

Porcine epidemic diarrhea virus (PEDV) and porcine rotavirus A (PoRVA), as primary pathogens of viral diarrhea in piglets, lead to substantial economic losses in global swine production. Existing detection approaches-including virus isolation, ELISA, and PCR-are limited by prolonged turnaround times, dependence on specialized equipment, or low sensitivity, which hinder their utility for rapid on-site diagnosis. In this study, we established RT-MIRA-LFD, a rapid, sensitive, and visually interpretable on-site detection platform for PEDV and PoRVA, leveraging multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD). Forward and reverse primers targeting conserved regions of the PEDV N and PoRVA VP6 genes were designed with 5'-6-FAM and 5'-biotin modifications, respectively, enabling seamless integration of MIRA amplification and LFD readout. Sensitivity testing revealed detection limits of 20 copies/μL (PEDV) and 4.5 copies/μL (PoRVA) for plasmid standards. Both assays showed no cross-reactivity with TGEV, PDCoV, CSFV, or other common swine pathogens, confirming high specificity. Clinical validation with 50 anal swab samples demonstrated 98 % (PEDV) and 96 % (PoRVA) concordance with RT-qPCR, supported by Kappa values of 0.92 and 0.96. The entire workflow completes within 30 min, bypasses complex instrumentation, and allows direct visual interpretation, dramatically enhancing field-deployable detection efficiency. This RT-MIRA-LFD platform offers a practical solution for early diagnosis, outbreak monitoring, and targeted control of PEDV and PoRVA, especially in resource-limited settings, thereby supporting efforts to reduce economic impacts on swine operations.