Development and Application of a Reverse Transcription Multienzyme Isothermal Rapid Amplification Assay Coupled with a Lateral Flow Dipstick for Rapid Detection of PEDV and TGEV

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are major causes of viral enteritis in piglets and produce overlapping clinical signs, complicating differential diagnosis in the field. Here, we developed singleplex and duplex visual assays combining reverse transcription multienzyme isothermal rapid amplification (RT-MIRA) with a lateral flow dipstick (LFD) for the detection and differentiation of PEDV and TGEV, targeting the conserved PEDV M gene and TGEV N gene. After screening the primer-probe sets, amplification was performed at 35 °C within 20 min, and the products were read visually on colloidal gold-based strips. No cross-reactivity was observed with reference standards of porcine deltacoronavirus (PDCoV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine rotavirus (PoRV), or porcine reproductive and respiratory syndrome virus (PRRSV). Preliminary analytical limits of detection were 2.1 × 101 copies/μL for PEDV and 2.0 × 101 copies/μL for TGEV (approximately 84 and 80 copies per reaction, respectively), as determined with in vitro-transcribed RNA standards. In 50 clinical fecal samples from suckling piglets at three farms in one geographic region, with RT-qPCR as the comparator, RT-MIRA-LFD correctly identified all six RT-qPCR-positive samples, comprising four PEDV single infections, one TGEV single infection, and one mixed infection, and yielded two single-target false-positive results, giving specificities of 97.8% (PEDV) and 97.9% (TGEV); these clinical results should be regarded as preliminary. These findings indicate that RT-MIRA-LFD may provide a rapid and practical tool for the differential detection of PEDV and TGEV in field settings. However, because the TGEV assay targets the N gene, it cannot differentiate TGEV from the closely related porcine respiratory coronavirus (PRCV).