## 1 Introduction
If the sample is positive, the ISO involves the use of selective-differential agar to determine whether the virulence factors are contained within a single STEC cell; otherwise, the sample is labeled as \"presumptive positive\" (i.e., samples with a mix of bacteria, each harboring only one of the targets). The main drawbacks of quantitative PCR (qPCR) are: the additional time of analysis required in the case of positive samples, the high limit of sample quantification (\(10^{3}\)-\(10^{4}\) CFU/g) in comparison with the culture-dependent techniques [35; 36; 37; 38] and the total dependence on the accuracy of standard curve construction [39]. During the last decade, a \"third-generation PCR\", or digital PCR (dPCR), has been developed as a technique for the sequence-specific detection and direct quantification of nucleic acids [40; 41]. This could replace qPCR in several applications, since it has some advantages, such as high precision [42], and reduced interference of PCR inhibitors [43] and does not require a calibration curve in order to estimate the copy number concentration of the target sequence [44]. Among the various digital PCRs, the droplet digital PCR (ddPCR) from Bio-Rad (Bio-Rad Laboratory, Hercules, California, USA) has been used in several applications. This technique utilizes the phase separation of a water-in-oil emulsion in order to distribute the sample into thousands of small-volume reaction chambers (partitions), thus enabling the positive and negative droplets within each partition to be counted. Specifically, the fluorescence signal of each droplet is individually counted. Through the application of the Poisson distribution, the fraction of positive droplets can be used to calculate the number of copies of the target gene without elaborating a standard curve [45; 46; 47]. The data yielded by the ddPCR droplet reader can be investigated according to four experimental types: absolute quantification (ABS), copy number variation (CNV), rare event detection (RED), and gene expression (GEX). The above different approaches make the ddPCR a valid instrument for several applications. Absolute quantification (ABS) allows the quantification of nucleic acids in copies per microliter of the sample, without requiring a standard curve. It has been used to detect several bacteria, parasites, and viruses [47; 48; 49; 50]. Copy number variation (CNV) shows the copy number variations of a \"target\" locus by comparing it with that of an invariant reference locus [51]. Recently, ddPCR has been used to determine CNV in pathogens and food [52; 53]. Gene expression studies (GEX) are carried out to provide information on the function of a gene. In this case, ddPCR is able to detect low levels of DNA gene expression even in the presence of inhibitors [54; 55]. Rare event detection (RED) can be grouped into two sub-applications: rare mutation detection (RMD) and rare sequence detection (RSD). Like ABS, these techniques are forms of direct quantification of nucleic acid target sequences in cancer research or virus studies [48]. The present study aimed to develop a new approach to the quantification of the main virulence genes of STEC in less time than that required by UNI CEN ISO/TS 13136: 2012. Hence, the proposed method of analysis could constitute an alternative to the conventional Real-time PCR and could act as a support for the above ISO.