Detection of Viable Vibrio cholerae Cells in Seafood Using a Real-Time Visual Loop-Mediated Isothermal Amplification Combined with Propidium Monoazide

Abstract

Vibrio cholerae is an important foodborne pathogen causing severe intestinal infectious diseases that have high incidence and mortality. Almost all of rapid testing methods including immunological and molecular assays for V. cholerae are incapable of distinguishing live cells from dead ones, which may overestimate the number of bacteria and result in many false positive results. To address the problems, live cell-specific dye such as propidium monoazide (PMA) is employed. The loop-mediated isothermal amplification (LAMP) assay is a nucleic acid amplification method that is fast, specific, and sensitive. In this study, we developed a real-time visual LAMP assay using PMA dye to detect thyA gene, thereby identifying viable V. cholerae cells. The results showed that only V. cholarae strains could be detected, and there was no cross-reaction with non-V. cholarae strains. Besides, the sensitivity of the PMA-LAMP assay was 1.1 × 102 CFU/mL and the entire reaction could be accomplished within 1 h. The sensitivity was on par with that of the PMA-qPCR assay. The detection limit in different artificially inoculated samples was 5 CFU/25 g materials for the tested pathogens. In the practical test, the PMA-LAMP assay performed well in comparison with PMA-qPCR and the culture method. Hence, PMA-LAMP assay can provide a highly effective and rapid approach for detecting viable V. cholerae.