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<break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break><break></break>A detection and discrimination system for five <italic>Escherichia coli</italic> pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic <italic>E. coli</italic> (CoSYPS Path <italic>E. coli</italic>). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of <italic>E. coli:</italic> shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) <italic>E. coli</italic>. The SYBR® Green qPCR assays target the <italic>uidA</italic>, <italic>ipaH</italic>, <italic>eae</italic>, <italic>aggR</italic>, <italic>aaiC</italic>, <italic>stx1</italic>, and <italic>stx2</italic> genes. <italic>uidA</italic> controls for <italic>E. coli</italic> presence and all the other genes are specific targets of <italic>E. coli</italic> pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path <italic>E. coli</italic> system was subsequently evaluated on four food matrices artificially contaminated with pathogenic <italic>E. coli.</italic> It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.<break></break> [ABSTRACT FROM AUTHOR]