title: Application of the modular approach to an in-house validation study of real-time PCR methods for the detection and serogroup determination of verocytotoxigenic Escherichia coli.
abstract: European Commission regulation 2073/2005 on the microbiological criteria for food requires that Escherichia coli is monitored as an indicator of hygienic conditions. Since verocytotoxigenic E. coli (VTEC) strains often cause food-borne infections by the consumption of raw food, the Biological Hazards (BIOHAZ) panel of the European Food Safety Authority (EFSA) recommended their monitoring in food as well. In particular, VTEC strains belonging to serogroups such as O26, O103, O111, O145, and O157 are known causative agents of several human outbreaks. Eight real-time PCR methods for the detection of E. coli toxin genes and their variants (stx(1), stx(2)), the intimin gene (eae), and five serogroup-specific genes have been proposed by the European Reference Laboratory for VTEC (EURL-VTEC) as a technical specification to the European Normalization Committee (CEN TC275/WG6). Here we applied a "modular approach" to the in-house validation of these PCR methods. The modular approach subdivides an analytical process into separate parts called "modules," which are independently validated based on method performance criteria for a limited set of critical parameters. For the VTEC real-time PCR module, the following parameters are being assessed: specificity, dynamic range, PCR efficiency, and limit of detection (LOD). This study describes the modular approach for the validation of PCR methods to be used in food microbiology, using single-target plasmids as positive controls and showing their applicability with food matrices.
authors.affiliation: European Commission Joint Research Centre, Institute for Health and Consumer Protection, Molecular Biology and Genomics Unit, Via Enrico Fermi 2749, I-21027 Ispra (VA), Italy.
authors.forename: Dafni-Maria
authors.initials: DM
authors.lastname: Kagkli
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Weber
authors.forename: Marc
authors.initials: M
authors.lastname: Van den Bulcke
authors.forename: Silvia
authors.initials: S
authors.lastname: Folloni
authors.forename: Rosangela
authors.initials: R
authors.lastname: Tozzoli
authors.forename: Stefano
authors.initials: S
authors.lastname: Morabito
authors.forename: Monica
authors.initials: M
authors.lastname: Ermolli
authors.forename: Laura
authors.initials: L
authors.lastname: Gribaldo
authors.forename: Guy
authors.initials: G
authors.lastname: Van den Eede
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D004927:Escherichia coli Infections / Q000517:prevention & control; D005516:Food Microbiology; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D054323:Shiga-Toxigenic Escherichia coli / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*