Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBRHGreen Real-Time PCR Methods and High Resolution Melting Analysis

Escherichia coli is a group of bacteria which has raised lots of concerns in the recent years. Five major intestinal pathogenic groups have been recognized among which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively). 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 foodborne diseases; it has also addressed the need for validated methods. High Resolution Melting analysis allows the discrimination among very similar sequences, with stx sequences being such an example. Therefore, we developed a real-time PCR flexible system 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, which was responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature, and they were further multiplexed so that they would demand less time and initial DNA material. Moreover, HRM analysis allowed for the discrimination among strains possessing different profiles, meaning different combination of traits. The development, application to food samples and the possible flexibility of the methods are thoroughly discussed. A decision support system based on the amplification and melting data has also been developed to support the detection.

KAGKLI Dafni;  FOLLONI Silvia;  BARBAU-PIEDNOIR Elodie;  VAN DEN EEDE Guy;  VAN DEN BULCKE Marc; 

2012-07-26

PUBLIC LIBRARY SCIENCE

JRC67799

1932-6203,   

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0039287,    https://publications.jrc.ec.europa.eu/repository/handle/JRC67799,   

10.1371/journal.pone.0039287,