Towards a pathogenic Escherichia coli detection platform using multiplex SYBR®Green Real-time PCR methods and high resolution melting analysis

Abstract

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.

Figure 1. Dynamic range of all methods.

Letters indicate significantly different values (p<0.05).

Figure 2. Melting peaks of amplicons in (A) triplex and (B) duplex PCR.

Respective targets are indicated on each graph.

Figure 3. HRM of reference strains: (A) Simplex reactions, (B) Duplex reactions.

Grey group: stx1+stx2+eae, blue: stx2+eae, red: stx1+eae, green: stx2, pink:stx1, (C) Triplex reations. Blue: stx1+stx2+eae, yellow: stx2+eae, pink: stx1+eae, green: stx1, red: stx2+aggR, grey: eae.