Development of Methods for Enrichment and PCR Detection of Important Non-O157 Shiga Toxin-Producing E. Coli

Project Methods:
This collaboration involves the inclusion of multiple aspects for the design and optimization of methods yielding successful detection of six serogroups of non-O157:H7 STECs, including O26, O45, O103, O111, O121, and O145. The first aspect provides focus on the effort to optimize enrichment methods used for successful propagation of all serogroups of interest. In regard to real-time PCR assay development, there will be two areas of focus. The first includes developmental efforts for the determination of the presence of Shiga toxin genes and the intimin gene (eae) as a screening method. The second area focuses on methods for the individual identification of each of the six non-O157:H7 serogroups listed above based on amplification of serogroup-specific genes. Upon optimization of the chemistry, inclusion of an internal positive control, and determination of grouping of serogroup specific primers and probes, the PCR reagents will be evaluated using reagents in the form of a tablet. Evaluation of the entire process of enrichment, detection of toxin and intimin genes, and identification of each STEC serogroup using the tableted PCR reagents, is the goal of this collaboration.