P3-154 Rapid Real-time PCR Method for Detection of Enterohemorrhagic Escherichia coli (EHEC) in Raw Romaine Lettuce

Wednesday, July 31, 2013
Exhibit Hall (Charlotte Convention Center)
Josephine Greve, Kwik Trip, Inc./University of Wisconsin-La Crosse, La Crosse, WI
Mark Zietlow, Kwik Trip, Inc., La Crosse, WI
Kevin Miller, Marshfield Food Safety, LLC, La Crosse, WI
Jay Ellingson, Kwik Trip, Inc., La Crosse, WI
Introduction: Enterohemorrhagic Escherichia coli (EHEC), including E. coli O157:H7, cause human illness worldwide via consumption of contaminated food and water. EHEC produce Shiga toxin, encoded by stx genes, and attachment and effacing lesions on human intestinal lining, encoded by eae gene. Several studies have used molecular-based methods to confirm the presence of eae gene in EHEC in bovine products and feces.

Purpose: Since beef is no longer the only food source of EHEC, this study focused on EHEC contamination of raw romaine lettuce from retail outlets. The purpose was to modify the methods from a previous study and apply them to EHEC testing on raw romaine lettuce.

Methods: Every other month (8/2010 – 12/2011), up to 9 lettuce heads were purchased from each of 9 different retail stores; 3 stores in each state: Iowa, Minnesota and Wisconsin. Samples were diluted (1:9), stomached (2 min, 230 rpm), enriched (8-16 h, 42°C), immunomagnetically separated with magnetic beads specific for E. coli O157:H7, and used for real-time PCR with both Roche LightCycler® (targeting eae gene) and Idaho R.A.P.I.D.® LT (targeting E. coli O157:H7). Method specificity and sensitivity were tested using mixed cultures of various percentages of EHEC strains, i.e., 25% of O157 with 75% O111.

Results: Of the 720 lettuce heads tested, only 2 (0.28%) were positive for eae gene, as indicated by melting peak presence. However, all samples were negative for E. coli O157:H7. This test method was shown to have a detection limit of 1-10 organisms per sample with and without an immunomagnetic separation step for O157:H7, O45 and O26 isolates.

Significance: No E. coli O157:H7 was found in raw romaine lettuce samples tested, so current preventative measures and test methods seem to be effective. This test method is specific and sensitive for the rapid and simultaneous detection of EHEC strains containing the eae gene.