## 2 Materials and Methods
### Droplet Digital PCR  
Droplet digital PCR was carried out by means of the Bio-Rad QX200 system (Bio-Rad Laboratory, Hercules, CA, USA). Briefly, the ddPCR reactions were carried out by using a reaction volume (20 uL) consisting of 50 ng of DNA, 10 uL ddPCR Supermix for Probes (No dUTP), 0.5 uM of each primer, 0.2 uM of the probe and nuclease-free water as required to obtain the final volume. Primers and Probes sequences are reported in Table 2.  
The reaction mixture was used to produce the droplet mix in a DGS cartridge (Bio-Rad Laboratory, Hercules, CA, USA) using the QX100 droplet generator (Bio-Rad Laboratory, Hercules, CA, USA). The emulsion (40 uL) of droplet-partitioned samples was then transferred to a 96-well plate and amplified by using a C1000 Touch Thermal Cycler (Bio-Rad Laboratory, Hercules, CA, USA) and adopting the following thermal profile: 95 \({}^{\circ}\)C for 10 min, 45 cycles consisting of 95 \({}^{\circ}\)C for 15 sec and 58.5 \({}^{\circ}\)C for 1 min, and a final step at 98 \({}^{\circ}\)C for 10 min. Subsequently, the 96-well plate was read by means of the QX200 Droplet Reader, in terms of the number of positive droplets and according to a Poisson distribution. QuantaSoft software was used to count the PCR-positive and PCR-negative droplets, in order to provide absolute quantification of the target DNA. The quantification measurements of each target were expressed as the number of copies per 1 uL/reaction. In  
\begin{table}
\begin{tabular}{c c} \hline
**Bacteria** & **Code (ATCC) \({}^{*}\)** \\ \hline _Salmonella enterica_ Enteritidis & 13076 \\ \hline _Salmonella enterica_ Typhimurium & 14028 \\ \hline _Campylobacter coli_ & 43478 \\ \hline _Campylobacter jejuni_ & 33291 \\ \hline _Escherichia coli_ & 25922 \\ \hline _Bacillus cereus_ & 11778 \\ \hline _Pseudomonas aeruginosa_ & 27853 \\ \hline _Shigella somei_ & 25931 \\ \hline _Staphylococcus aureus_ & 25923 \\ \hline _Yersinia enterocolitica_ & 9610 \\ \hline \end{tabular} \({}^{*}\)American Type Culture Collection. After incubation at 37 \({}^{\circ}\)C for different times, all the samples were subjected to a DNA extraction process.  
\end{table}
Table 1: List of bacteria selected for the analyses of specificity and exclusiveness.  
\begin{table}
\begin{tabular}{c c} \hline
**Primer Name** & **Sequence** \\ \hline STX1 (Fw) & TITGTYACTGSTASCAGCWGAAGCYTTACG \\ STX1 (Rev) & CCCCAGTTCARWGTAGRTCMACRTC \\ STX1 (Probe) & FAM \({}^{1}\)—CTGGATGATCTCAGTGGGCGCTTATGTA\({}^{2}\) \\ \hline STX2 (Fw) & TITGTYACTCTSACACWGAAGCYTACG \\ STX2 (Rev) & CCCCAGTTCARWGTAGRTCMACRTC \\ STX2 (Probe) & FAM—TCGTCAGGCACCTGTCTGAACCTCTCC—BHQ \({}^{3}\) \\ \hline eae (Fw) & CATTGATCAGGATTTTTCTGGTA \\ eae (Rev) & CTCATGCGGAAAATAGCCGTTA \\ eae (Probe) & FAM—ATAGTTCCGCCGATATTGCCGACACACATAC—TAMRA \\ \hline \end{tabular} \({}^{*}\)Notes: \({}^{1}\) FAM: 6-Carboxylfluorescein; \({}^{2}\) TAMRA: 5-Carboxytetramethylhydndamine; \({}^{3}\) BBQ: black hole quencher.  
\end{table}
Table 2: Sequences of primers and probes used in Real-Time PCR and digital droplet PCR.  
addition, in the case of a saturation condition, several dilutions of the samples were carried out up to 10\({}^{-9}\).