TY - JOUR T1 - Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR(R)Green Real-Time PCR Methods and High Resolution Melting Analysis418 JF - PLoS.One. Y1 - 2012 A1 - Kagkli,D.M. A1 - Folloni,S. A1 - E. Barbau-Piednoir A1 - Van den Eede,G. A1 - Marc Van den Bulcke KW - a KW - ALL KW - analysi KW - analysis KW - application KW - article KW - at KW - Attention KW - bacteria KW - Biology KW - Case KW - consumer KW - detection KW - detection method KW - developing KW - Development KW - discrimination KW - disease KW - Diseases KW - dye KW - e KW - electronic KW - Escherichia coli KW - European KW - European Commission KW - food KW - Food Safety KW - gene KW - Genes KW - genomic KW - Genomics KW - Germany KW - Group KW - health KW - HRM KW - im KW - Institute KW - IS KW - ITALY KW - journal KW - method KW - methods KW - Molecular KW - Molecular biology KW - Monitoring KW - need KW - Order KW - outbreak KW - outbreaks KW - pathogenic KW - PCR KW - protection KW - protein KW - real time PCR KW - Real-time PCR KW - report KW - Research KW - SAFETY KW - Sample KW - Samples KW - SB - IM KW - Shiga Toxin KW - STEC KW - strain KW - SYBR(r)Green KW - Temperature KW - toxin KW - Traits KW - use KW - virulence KW - VTEC AB - Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the "top-five" serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform VL - 7 CP - 6 U1 - 418 M3 - 10.1371/journal.pone.0039287 [doi];PONE-D-12-04306 [pii] ER -