## 1 Introduction  
Vercorytotoxin-producing _Escherichia coli_ (VTEC)--also called Shiga-toxin-producing _Escherichia coli_ (STEC)--are zoonotic agents that can cause several human illnesses, such as diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) [1]. STEC infections are continuously growing and are the third most commonly reported zoonosis in the EU. In 2018, 8161 confirmed cases of human STEC infections occurred in the EU, mostly in Ireland, Sweden, Malta, and Denmark, with an increase of 39% from 2017 [2]. The bacteria can be transmitted to humans through person-to-person contact, consumption of food or water contaminated with animal feces, or by direct contact with animals [3, 4]. Data reported by the EPSA over the period 2014-2018, revealed that beef and its derivatives continued to be the main vehicles of human STEC infections [2]. Furthermore, the easy transmission and very low infectious dose (i.e., <10 cells) of this bacterium make it able to induce both outbreaks and sporadic cases of human disease [5]. In addition to verocytotoxin (_vtx/stx_) genes, another virulence factor is the protein \"intimin\", which is encoded by the \"_eae_\" gene. Intimin is involved in the attachment of STEC bacteria to the intestinal epithelial cells [6]. Since _E. coli_ O157:H7 represent one of the most common causes of STEC infections [7], the simplest classification of STEC bacteria consists of _E. coli_ O157 and non-O157 STEC (i.e., O26, O91, O103, O104, O111, O117, O128, etc) [8, 9]. The rate of STEC infection rises markedly during the summer period, owing to several factors: greater consumption of barbecued hamburgers, the shedding of STEC by cattle, a higher bacterial load in minced beef, increased environmental contamination, and longer survival of the bacteria at the time of slaughter [10; 11]. In particular, the contamination of carcasses with STEC bacteria can occur when gut contents or fecal matter come into direct contact with meat surfaces or by cross-contamination through contact with contaminated carcasses or environments [12; 13]. In addition, cattle are the main reservoir of STEC strains, and foodstuffs of beef origin are the main sources of human infection [10; 14]. To date, there are few data on STEC contamination of beef products in Italy [15; 16; 17], as the surveillance of _E. coli_ O157 and non-O157 in meat or other products is not included in European or national legislation [18; 19]. The clinical and economic consequences of the spread of STEC have prompted the development of various methods for detecting STEC strains, which are alternatives to the gold standard (bacteriological method) consisting of the use of selective media or chromogenic agar. Its limits are: the time required for the response (several days), the difficulty of interpreting the final results, due to the abundant background flora [20; 21; 22] or when the STEC are below the detectable limits of the test [23], the high labor commitment required for processing large numbers of samples [24] and the presence of commonly used antimicrobials, such as cefixime or tellurite, which may inhibit some STEC bacteria [23]. Given the above limitations, new approaches, such as those based on PCR [25; 26; 27], microarray [27; 28; 29], or whole genomic sequencing [27; 30; 31], have been proposed. Of these molecular methods, Real-time PCR, which enables one or more \"target\" genes to be identified either from one template [21] or multiple templates [26], is frequently used, owing to its differentiation potential and reliability [32; 33]. This method is carried out according to UNI CEN ISO/TS 13136: 2012 [34], which describes the identification of STEC through the detection of its main virulence genes. Specifically, the above ISO envisions the use of Real-time PCR as a tool for screening specific genetic markers, such as \"_eae_\" and \"_stx_\" genes.