Abstract: This study aimed to establish a duplex droplet digital polymerase chain reaction (ddPCR) assay for the rapid quantification of Cronobacter sakazakii and Listeria monocytogenes. Species-specific primers and probes targeting the ompA gene of C. sakazakii and the plcB gene of L. monocytogenes were designed. After systematic optimization of the reaction conditions, the analytical sensitivity, specificity and repeatability of the duplex ddPCR assay were evaluated, and the assay was subsequently applied to detect pathogens in 80 real samples. The optimal reaction conditions were annealing temperature 56 ℃, primer concentration 600 nmol/L, and probe concentration 350 nmol/L for C. sakazakii and 500 nmol/L for L. monocytogenes. No cross-reactivity was observed with 20 common foodborne bacteria, indicating high specificity. The inter- and intra-assay coefficients of variation were both < 16%, demonstrating excellent repeatability. The limits of detection (LOD) were 7.51 copies/reaction for C. sakazakii and 8.02 copies/reaction for L. monocytogenes, which were significantly superior to those of conventional qPCR. Our duplex ddPCR assay offers several advantages such as high specificity and stability, providing technical support for the absolute quantification of these two important foodborne pathogens.

Key words: Cronobacter sakazakii; Listeria monocytogenes; duplex droplet digital polymerase chain reaction; quantitative detection