Abstract：Food-borne microorganisms are important factors affecting food quality and safety. Droplet digital PCR (ddPCR), which can be used for the absolute quantitative detection of nucleic acids, plays an increasingly important role in the detection of food-borne pathogens. In order to quantify the genome copy numbers of food-borne pathogens, a strategy for simultaneous detection of the DNA copy numbers of Salmonella typhi, Staphylococcus aureus and Listeria monocytogenes by multiplex droplet digital PCR (ddPCR) was developed in this study. Primers and probes targeting ttrA/ttrC、FMN-binding glutamate synthase、invasion associated endopeptidase gene were selected to evaluate the feasibility and applicability of the strategy by specific verification using TaqMan probe qPCR. The probes for Salmonella typhi and Staphylococcus aureus were labeled with 6-carboxy-fluorescein (FAM) fluorophore, and the probe for L. monocytogenes was labeled with hexachloro fluorescein (HEX) fluorophore. The detection systems of simplex ddPCR for 3 target strains were constructed. On this basis, through the optimization of primer and probe concentration and annealing temperature, a multiplex ddPCR detection system for simultaneous, rapid and absolute quantitative detection of genomic copy numbers of Salmonella typhi, Staphylococcus aureus and L. monocytogenes in the same reaction system was constructed. The multiplex ddPCR detection system could obtain fluorescent amplification with 8 (23) distinct clusters under the optimal condition obtained by means of annealing temperature optimization, and the quantitative linear range of these 3 pathogens were 235~0.23、284~0.18、380~0.42 copies/µL, which were almost the same as simplex ddPCR for single strain, and the linear correlation coefficient R2 in the quantitative detection range were all greater than 0.999 with excellent repeatability. The constructed multiplex ddPCR detection system had strong specificity, good stability and wide quantitative range, which could provide technical support for absolute quantitative detection of food-borne pathogens without cultivation.
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