Innovative Phage Protein Ligand Automated Assay to Simplify the Confirmation of TOP 7 EHEC in Raw Beef

Introduction: The MLG 5B reference method (USDA) recommends an Immuno Magnetic Separation (IMS) for selective concentration of the TOP 7 EHEC serogroups from pre-enriched samples to assist in plate isolation step. However, this technique is cumbersome and difficult to implement routinely. The automated VIDAS^® UP E. coli Serogroups (ESPT) assay, a specific phage protein ligand assay, has been developed as an alternative to IMS for the simultaneous immuno-concentration of E. coli serogroups O26, O45, O103, O111, O121, O145 and O157.

Purpose: To compare the VIDAS ESPT assay with a commercial IMS test for the confirmation of TOP 7 EHEC in 375-g sample size of raw beef.

Methods: 375g artificially inoculated samples, ¼ diluted in a selective pre-warmed Buffered Peptone Water, were enriched for 10 hours at 41.5±1°C and then screened by PCR for the presence of eae, stx and O groups genes. All presumptive positives were isolated on the chromID^®EHEC agar following the VIDAS ESPT and the IMS procedures. Characteristic colonies were further confirmed according to the MLG 5B method.

Results: In this comparative study, 100% of the presumptive PCR positive were confirmed with the new method compared to 86% with the IMS method. Furthermore, in 86% of the cases, the analysis of a single typical colony was sufficient to confirm the presumptive results by using the VIDAS method. For three presumptive positive samples, the analysis of up to ten colonies obtained after IMS did not allow confirmation of the initial PCR results.

Significance: Due to high level of specificity of the recombinant phage protein, the novel assay significantly reduced the background flora on the selective agar and facilitated the recovery of the specific strains. Compared with the traditional IMS, the VIDAS ESPT greatly reduced the number of colonies for subsequent confirmation and improved the overall confirmation rate of the TOP-7-EHEC.