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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.Microbial analysis of foodstuffs is an integrated part of the management of microbial safety in the food chain. Standardized detection methods exist, which are based on the growth and isolation of the microorganism of interest and are in general referred to as "classical" culture methods. Based on the general expertise and supported by an international consensus in the field of food microbiology, the current detection methods are harmonized and commonly recognized as the standard detection methods (17). These methods are considered precise, practical, and relatively inexpensive in terms of laboratory consumables and reagents but are rather time-consuming; 3 to 7 days may be required before isolation and characterization, e.g., serotyping of the pathogen, are obtained. Currently, standardized methods exist for the detection of Escherichia coli O157 but not for the top five verocytotoxigenic E. coli (VTEC) serogroups; therefore, the scientific opinion of the European Food Safety Authority (EFSA) panel on Biological Hazards (BIOHAZ) strongly recommended the development and validation of methods for the detection of the top five VTEC serogroups: O26, O103, O111, O145, and O157 (6, 7).During the last decade, several food-borne outbreaks have been reported, e.g., the recent emergency of the E. coli O104:H4 outbreak, and a strong demand for "rapid methods" has risen. The term "rapid" refers especially to the need for a short delay between an emergency alarm due to a potential pathogen outbreak and the outcome an...

An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.