Abstract:[Objective] A detection method for viable Listeria monocytogenes (LM) was established combining improved propidium monoazide (PMAxx) with droplet digital PCR (ddPCR). [Method] After optimization of the final PMAxx concentration, dark treatment time, and exposure time, the optimal PMAxx-ddPCR detection system was established. Different volume ratios of viable LM were employed to verify the accuracy of the PMAxx-ddPCR method, and the specificity, sensitivity, and reproducibility of this method were examined. Cheese stick samples were artificially contaminated with different concentrations of viable and dead LM and then used to compare the detection results of the conventional plate counting method and the PMAxx-ddPCR method established in this study. Additionally, commercially available enoki mushrooms and ham sausages were tested to evaluate the practical detection performance of the PMAxx-ddPCR method. [Result] At a final PMAxx concentration of 10 μmol/L, dark treatment time of 10 min, and exposure time of 10 min, PMAxx significantly inhibited the DNA amplification of dead bacteria, while having no significant effect on that of viable bacteria. The developed PMAxx-ddPCR method only generated specific amplification for LM, with a limit of quantification of 147 CFU/mL. The relative standard deviations (RSD) of the method were consistently less than 20%, indicating good repeatability. In the artificial contamination experiment of cheese sticks, the quantification results of LM by the plate counting method and the PMAxx-ddPCR method had no significant difference (P>0.05). Further detection of 17 enoki mushroom samples and 16 ham sausage samples revealed that LM was detected in 7 enoki mushroom samples by both PMAxx-ddPCR and plate counting methods. Moreover, the quantitative results of the two methods showed no significant difference (P>0.05). [Conclusion] A PMAxx-ddPCR method is established for detecting viable LM, enabling specific quantitative detection of viable LM in food.
