Real-time loop-mediated isothermal amplification of propidium monoazide for visualization of viable Bacillus cereus in food

Bacillus cereus is a common contaminating bacteria and conditioned pathogens in food. Immunological methods or molecular biology assays that are currently available cannot distinguish between live and dead bacterial cells, which may overestimate the number of bacteria and lead to false-positive results. To solve this problem, we introduced propidium monoazide (PMA) and calcein in this study and established a real-time visual loop-mediated isothermal amplification (LAMP) detection method to detect viable B. cereus. In addition, the results can be detected visually with calcein dye or quantitatively by monitoring the amplification curve of the fluorescence signal. In this study, the results showed that the visual PMA-LAMP method had high specificity, strong anti-interference ability, a sensitivity of 1.16 x 10(2) CFU/mL, and that the whole reaction could be completed within 80 min. This method not only has high sensitivity, but also has a shorter reaction time and is faster. In the detection of artificially contaminated food samples, the lowest detection limit reached 6 CFU/mL after 3 or 4 h of enrichment. Therefore, the visual PMA-LAMP technology can provide an efficient and rapid method for the detection of live B. cereus bacteria.