Edan Hosking

11:05-11:20 a.m., Tuesday
Edan Hosking, PhD
Senior Research Scientist
R&D, Molecular Platform Development
Neogen Corporation

620 Lesher Pl., Lansing, MI 48912

NeoSEEK: EHEC/STEC detection and identification via genotypic profiling

Enterohemorrhagic Escherichia coli (EHEC) are recognized as the primary cause of hemorrhagic colitis or bloody diarrhea. EHEC, a subset of STEC, are comprised of pathogenic strains, including the prototypic serotype O157:H7 but also numerous other serotypes that have caused illness worldwide (e.g., O26, O45, O103, O111, O121, O145). We have adapted the Sequenom® high throughput SNP genotyping platform to develop a rapid molecular method to detect the presence and identity of STEC in a complex sample.

The Sequenom platform relies on MALDI-TOF Mass Spec-based multiplexing. Specifically, this platform has three steps: 1) PCR amplification; 2) Primer extension to generate allele-specific DNA products of different masses; and 3) Chip-based mass spec to analyze the extension products. The test is performed by establishing the presence/absence pattern of target genes based on “molecular profiles” developed for the 7 STEC of interest. A total of 70 targets are assayed, including targets for O group, H type, stx, several virulence associated genes and subtypes, and 8 specific SNP targets to assist in STEC/non-STEC differentiation. The number and types of targets were selected to gather enough evidence to determine if a detected O group is an STEC without the need for single colony isolation.

A validation study was performed as part of an application for a Letter of No Objection from USDA-FSIS. An independent testing laboratory performed 49 inoculation events, each with a low and high level in 375-g beef trim samples. Samples were blind coded and tested by the NeoSEEK method and by the USDA-FSIS reference method. In the low spike samples, there were 17 and 15 STEC positives by the NeoSEEK and reference methods, respectively. For the high spike samples, there were 31 and 28 STEC positives by the NeoSEEK and reference methods, respectively.  There was evidence of 5 false-negative results by the reference method in this study. Neither method produced a false-positive result.

Detection of O group(s), and STEC/non-STEC determination of such, can be made from an isolate or complex enrichment sample in a simple and economical assay for the 7 STEC of current concern in the U.S. From a presumptive positive enrichment culture or isolated colony, identifications can be made in less than 24 hours. As NeoSEEK is an “open platform”, additional targets can be added as need arises and therefore the test can be customized for research purposes, confirmation/identification, outbreak investigation, prevalence studies, and risk assessment.