Development of propidium monoazide-droplet digital PCR for Listeria monocytogenes quantification and growth modeling in ice cream

Listeria monocytogenes is a foodborne pathogen widely distributed in various food matrices and poses a serious health risk to susceptible populations. This study developed a quantitative detection method for viable L. monocytogenes by combining propidium monoazide (PMA) treatment with droplet digital PCR (ddPCR), and evaluated its application in ice cream and predictive growth modeling. Primers and probes targeting the hlyA gene showed high specificity, and the PMA-ddPCR assay maintained good anti-interference performance in the presence of high concentrations of background bacteria. The limit of detection (LOD) of the assay in ice cream was 1.0 × 10 2 colony-forming units/mL. Among 500 commercially available ice cream samples tested using the developed PMA-ddPCR assay, two samples were positive for L. monocytogenes , with copy numbers of 7.25 and 40.6 copies/µL, respectively. Mathematical models based on PMA-ddPCR and plate count results were established to characterize the growth behavior of L. monocytogenes in ice cream. The results obtained by the two methods under isothermal and fluctuating temperature conditions were highly consistent. The primary, secondary, and dynamic models all showed satisfactory fitting performance for describing the growth curves generated by both analytical methods. In conclusion, the PMA-ddPCR assay provides a viability-selective and culture-independent approach for the absolute quantification of L. monocytogenes in the tested ice cream matrix and may serve as a complementary molecular data source for microbial predictive modeling. However, appropriate matrix-specific validation is necessary.