Simple SummaryPorcine epidemic diarrhea virus (PEDV), porcine bocavirus (PBoV), and porcine rotavirus (PoRV) are associated with porcine viral diarrhea. The three viruses are often comorbid in mixed infection; therefore, it is necessary to develop a specific and rapid method for simultaneous detection. In this study, a multiple detection method, loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD), was established to detect three pathogens in 125 field samples. The consistency between the real-time fluorescence quantitative PCR (rt-qPCR) and the triplex LAMP–LFD assay was over 99%. The assay has been proven to be highly specific and sensitive.AbstractPorcine epidemic diarrhea virus (PEDV), porcine bocavirus (PBoV), and porcine rotavirus (PoRV) are associated with porcine viral diarrhea. In this study, triplex loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD) was established for the simultaneous detection of PEDV, PoRV, and PBoV. The PEDV-gp6, PoRV-vp6, and PBoV-vp1genes were selected to design LAMP primers. The amplification could be carried out at 64 °C using a miniature metal bath within 30 min. The triplex LAMP–LFD assay exhibited no cross-reactions with other porcine pathogens. The limits of detection (LODs) of PEDV, PoRV, and PBoV were 2.40 × 101copies/μL, 2.89 × 101copies/μL, and 2.52 × 101copies/μL, respectively. The consistency between rt-qPCR and the triplex LAMP–LFD was over 99% in field samples testing. In general, the triplex LAMP–LFD assay was suitable for the rapid and simultaneous detection of the three viruses in the field.