Triplex-Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Immunoassay for the Simultaneous Detection of Three Pathogens of Porcine Viral Diarrhea Syndrome in Swine

SIMPLE SUMMARY: Porcine 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. ABSTRACT: Porcine 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-vp1 genes 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 × 10(1) copies/μL, 2.89 × 10(1) copies/μL, and 2.52 × 10(1) copies/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.