Identification of Escherichia Coli Verotoxins by the Meridian Premier EHEC Kit®

Laboratory Procedure MFLP-93
November 2005

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Don Warburton
Evaluation Division
Bureau of Microbial Hazards, Food Directorate
Postal Locator: 2204A1
Ottawa, Ontario, K1A OL2

E-mail: Don_Warburton@hc-sc.gc.ca

1. Application

The Premier EHEC test is a rapid in vitro microwell EIA for the detection of Shiga verotoxins, or Shiga-like toxins I and II (8.1 - 8.10), in food or food ingredients to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. This test kit may also be used to verify verotoxin production of cultures isolated from samples analyzed using Laboratory Procedures MFLP-80 (or equivalent). This revised method replaces MFLP-93, dated April 1997.

2. Principle

The Premier EHEC test utilizes monoclonal anti-verotoxin capture antibody absorbed to microwells. Diluted samples are added to the wells and incubated at room temperature. A wash is performed to remove unbound material. A polyclonal anti-verotoxin antibody is added for detection and incubated at room temperature. Another wash is used to remove unbound antibody. Enzyme conjugated anti-IgG polyclonal antibody is added and incubated at room temperature. If toxin is present, a reactive antibody-enzyme complex is formed. After washing to remove unbound conjugate, substrate is added and incubated for ten minutes at room temperature. Color develops in the presence of bound enzyme. Stop solution is added and the results are interpreted spectrophotometrically.

®The Meridian Premier EHEC kit is a registered trademark of Meridian Diagnostics, Inc., Cincinnati, Ohio.

3. Definition of Terms

See Appendix A of Volume 3.

4. Collection of Samples

See Appendix B of Volume 3.

5. Materials and Special Equipment

The reagents and components of the Premier EHEC kit is provided in one kit containing sufficient materials for 96 tests. The kit is available from Oxoid Inc., 217 Colonnade Road, Nepean, Ontario, K2E 7K3 (Tel: 1-800-267-6391).

6. Procedure

All other specific information related to either materials, equipment, general procedures, and interpretation of reults can be found in the documentation accompanying each individual test kit.

7. Interpretation of Results

Follow the manufacturer's instructions in the accompanying documentation in each individual kit.

8. References

8.1 Acheson, D.W.K., Lincicoma, L.L., DeBreucker, S., and S.T. Keusch. 1996 Detection of Shiga-Like Toxin-Producing Escherichia coli in Ground Beef and Milk by Commercial Enzyme Immunoassay. J. Food Prot. 59:344-349

8.2 Griffin, P.M., Ostroff, S.M., Tauxe, R.V., Green, K.D., Wells, J.G., Lewis, J.H. and P.A. Blake. 1988. Illnesses associated with Escherichia coli 0157:H7 infections. A broad clinical spectrum. Ann. Intern. Med. 109:705-712.

8.3 Karmali, M.B. 1987. Laboratory diagnosis of verotoxin- producing Escherichia coli infections. Clin. Micro. Newsletter. 9:65-70.

8.4 Kay, B.A., Griffin, P.M., Stockbine, V.A. and J.G. Wells. 1994. Too fast food: Bloody diarrhea and death from Escherichia coli 0157:H7. Clin. Micro. Newsletter. 16:17-19.

8.5 Mariani-Kurkdjian, P., Deamur, E., Milan, A., Picard, B., et.al. 1993. Identification of a clone of Escherichia coli 0103:H2 as a potential agent of hemolytic-uremic syndrome in France. J. Clin. Micro. 31:296-301.

8.6 Maniar, A.C., Williams, T., Anand, C.M. and G.W. Hammond. 1990. Detection of verotoxin in stool specimens. J. Clin. Micro. 28:134-135.

8.7 Neill, M.A. 1991. Escherichia coli 0157:H7. A pathogen of no small renown. Infec. Dis. Newsletter. 10:19-24.

8.8 O'Brien, A.D. and R.K. Holmes. 1987. Shiga and shiga-like toxins. Micro. Reviews. 51:206-219.

8.9 Read, S.C., C.L. Gyles, R.C. Clarke, H. Lior, and S. McEwen. 1990. Prevalence of verocytotoxigenic Escherichia coli in ground beef, pork and chicken in southwestern Ontario. Epidemiol. Infect. 105:11-20.

8.10 Willshaw, G.A., H.R. Smith, D. Roberts, J. Thirlwell, T. Cheasty, and B. Rowe. 1993. Examination of raw beef products for the presence of Vero cytotoxin producing Escherichia coli, particularly those of serogroup 0157. J. Appl. Bacteriol. 75:420-426.