## 3 Results  
The slices of beef were artificially contaminated with different concentrations of _E. coli_ O157:H7 and these data were confirmed by microbiological analysis (data not shown). The data yielded by the ddPCR analyses allowed us to identify the minimum incubation time for STEC detection. The ddPCR method was able to quantify the number of copies/\(\upmu\)L--for all the genes--after an incubation time of 7 h, in the case of slices of beef contaminated artificially with about 150 CFU/mL (Table 3). However, at lower levels of contamination (15 and 1.5 CFU/mL), STEC detection required 8 and 9 h of incubation at 37 \({}^{\circ}\)C, respectively (Table 3). On the basis of these data, we can conclude that a 9 h incubation at 37 \({}^{\circ}\)C is needed in order to detect a level of contamination of about 1.5 CFU/mL. After 11 h of incubation, a saturation condition was reached; this condition did not allow the quantification of STEC by ddPCR, but it did confirm the presence of bacteria. All these results revealed that the incubation time varied according to the initial level of contamination, but was less than the 18 h required by UNI CEN ISO/TS 13136: 2012. However, it is important to point out that qPCR was unable to detect STEC at lower concentrations, at the above times of incubation (7 or 11 h; data not shown). Moreover, it should be noted that there was an increase of about 1 log in the number of copies/\(\upmu\)L when incubation was prolonged by an hour, for all the concentrations selected (Table 3). The artificially contaminated slices of beef were also analyzed by means of Real-time PCR according to UNI CEN ISO/TS 13136: 2012. The results showed that STEC bacteria were significantly detected only after 18 h of incubation of the sample at 37 \({}^{\circ}\)C. Indeed, the \(\upDelta\)Ct values detected in samples incubated for less than 18 h were not statistically significant (\(p\)-value \(>\) 0.05), thus confirming the need for prolonged incubation (data not shown). \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & \multicolumn{3}{c}{**150 CFU**} & \multicolumn{3}{c}{**15 CFU**} & \multicolumn{3}{c}{**1.5 CFU**} \\ \hline
**Incubation** & **strx1** & **strx2** & **eae** & **strx1** & **strx2** & **eae** & **strx1** & **strx2** & **eae** \\ \hline T6 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 \\ T7 & 1.1 & 3 & 4.1 & 0 & 0.23 & 0.12 & 0 & 0 & 0 \\ T8 & 94 & 86 & 248 & 17 & 13.4 & 25 & 2.3 & 3.6 & 8 \\ T9 & 1159 & 898 & 2380 & 147 & 199 & 455 & 39.7 & 32.6 & 52.9 \\ T10 & no call 1 & no call & no call & 1382 & 1553 & 4050 & 392 & 392 & 1180 \\ T11 & no call & no call & no call & no call & no call & no call & 3910 & 2640 & 5900 \\ T12 & no call & no call & no call & no call & no call & no call & no call & no call \\ \hline \hline \end{tabular} Notes: 1 no call: saturation condition.  
\end{table} 
Table 3: Number of copies/\(\upmu\)L detected by droplet digital PCR in sponges artificially contaminated with different concentrations of _E. coli_ O157:H7 (150, 15, and 1.5 CFU/mL) and incubated at 37 \({}^{\circ}\)C for different times. When the same samples were analyzed by means of ddPCR, a saturation condition was observed. To overcome this problem, a second experiment was conducted. The results showed that the samples, independently from the concentration, needed to be diluted up to \(10^{-5}\) in order to be quantified by ddPCR (Table 4). The same situation was also observed even when the incubation time was 12 h.  
Furthermore, the results yielded by the ddPCR of the 10 slices of beef contaminated with different bacteria, listed in Table 1, showed the specificity and exclusiveness of the proposed approach (data not shown). The last phase was carried out to verify the ability of ddPCR to detect STEC bacteria in sponges used for the control of beef carcass at the time of slaughter, and to compare this proposed approach with the conventionally applied Real-Time PCR.