A Droplet Digital PCR Method for Simultaneous Detection and Quantification of S. aureus, L. monocytogenes, C. sakazakii, and M. bovis in Dairy Products

Foodborne bacterial pathogens, including S. aureus, L. monocytogenes, C. sakazakii, and M. bovis, pose significant threats to dairy safety and public health. Current detection methods, such as culture-based techniques and real-time quantitative PCR (qPCR), are either time-consuming or limited in absolute quantification accuracy. Herein, we developed and validated a novel quadruplex droplet digital PCR (ddPCR) assay for simultaneous detection and absolute quantification of these four pathogens in dairy products. The assay targets the femA, hly, ompA, and esxA genes, respectively, with optimized primer/probe concentrations of 500 nM/400 nM and an annealing temperature of 58 °C. The established method demonstrated high specificity, with no cross-reactivity against common dairy-associated bacteria. The limits of detection (LoDs) ranged from 7.04 to 10.31 copies/reaction, with coefficients of variation (CVs) below 14% for intra-assay and 10% for inter-assay repeatability. Notably, the ddPCR assay detected a 25% co-contamination rate compared to 13% by qPCR among 120 dairy samples, suggesting higher sensitivity for low-abundance targets. This quadruplex ddPCR platform offers a rapid, sensitive, and high-throughput solution for food safety surveillance, particularly in high-risk dairy matrices such as infant formula.