DETECTION OF ENTEROHEMORRHAGIC E. COLI (EHEC) IN FOOD PRODUCTS AND FOOD INGREDIENTS BY THE VIP FOR EHEC METHOD

Laboratory Procedure MFLP-87
June 2005

---

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 method is applicable to the detection of enterohemorrhagic E. coli 0157 from food products, food ingredients and environmental samples to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. The VIP device can be used for initial screening of all foods and samples that are analysed following the procedures outlined in MFLP-80.

This revised method replaces MFLP-87, dated September 1996.

2. DESCRIPTION

VIP for EHEC is a visual immunoprecipitate assay that detects enterohemorrhagic E. coli including E. coli O157:H7. It employs highly specific antibodies directed against EHEC antigens and has been specifically formulated to minimize cross-reactivity with many Enterobacteriaceae while maintaining superior sensitivity. The method has been shown to produce satisfactory results with contaminated foods in AOAC and HPB studies. This method can be used for the detection of EHEC in food products, food ingredients, and environmental samples.

3. PRINCIPLE

The VIP for EHEC method uses a proprietary reagent system to form an antigen-antibody- chromogen complex if EHEC is present. This ensures a high level of sensitivity and specificity to E. coli O157:H7. Appropriately enriched samples are added to the VIP unit; any EHEC antigens will bind to the antibody-chromogen complex as it flows across a supporting membrane. When EHEC is present, the antigen-antibody-chromogen complex will form a detection line in the test sample window. Sample flow will continue down the membrane to form a control line in the test verification window regardless of whether the sample contains EHEC.

4. DEFINITION OF TERMS

See Appendix A of Volume 3.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 3.

6. MATERIALS AND SPECIAL EQUIPMENT

1. VIP for EHEC ( BioControl Systems Inc., phone: (425) 603-1123, (800) 245-0113, FAX: (425) 603-0070, website: http://www.biocontrolsys.com.
   
   
2. Modified Trypticase Soy Broth and Novobiocin (mTSB-n) (See MFLP-80)
   
   
3. Colworth Stomacher 400, blender or equivalent
   
   
4. Incubators capable of maintaining 35°- 37° C and 42° C
   
   
5. Stomacher bags, with or without mesh inner bag, as appropriate
   
   
6. Filter tips for pipettes (See MFLP-80)
   
   
7. Triton X
   
   
8. Control cultures (use ATCC cultures or equivalent)
   positive control: E. coli O157:H7 or E. coli O157:NM (or other serovars of E. coli O157)
   negative control: E. coli (not O157)

7. PROCEDURE

Each sample unit may be analyzed individually or the analytical units may be combined. Carry out the test in accordance with the following instructions:

7.1 Handling of Sample Units:

7.1.1 In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.

7.1.2 Analyze sample units as soon as possible after their receipt in the laboratory.

7.2 Preparation for Analysis

7.2.1 Have ready sterile mTSB-n that has been pre-warmed to 35°C.

7.2.2 Clean the surface of the working area with a suitable disinfectant.

7.3 Preparation of Sample

7.3.1 To ensure a truly representative analytical unit, agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. To reduce the workload, the analytical units may be combined for analysis. It is recommended that a composite contain not more than 500 g.

7.3.2 Prepare a 1:10 dilution of the food by aseptically blending or stomaching 25 g or mL (the analytical unit) into 225 mL of the mTSB-n broth. Some spices have to be analyzed using a higher dilution (see MFLP 80).

7.3.3 For viscous samples (e.g., powdered cheese), or for other samples as required, add ≤ 2.25 mL of steamed Triton X-100 per 225 mL mTSB + n prior to incubation.

7.3.4 Incubate overnight (at least 18 hrs) at 42°C. For commodities expected to have a high number of competitive bacteria, such as ground beef and sprouts, incubation at 42°C has been shown to reduce the number of false-positive and negative reactions. It is recommended that larger analytical units greater than 25 g be incubated for 22 to 24 hours (see MFLP-80).

7.3.5 After incubation, retain the enriched broths under refrigeration at 2-8°C, for confirmation of presumptive positive samples.

7.4 VIP Assay Procedure

7.4.1 Open sealed pouch containing VIP units and remove required number of tests. One device is necessary for each test sample. VIP units may not be reused. Be certain to immediately reseal unused VIP units in pouch containing desiccant. Store at ambient temperature in a cool, dark location.

7.4.2 Gently shake enrichment broth, then allow food particles to settle. Enriched broths should be equilibrated to 25 to 37°C prior to running the test, especially if the enrichment broth has been incubated at 42°C or refrigerated, as 25-37°C is the optimum temperature for the VIP test. It is recommended that samples are removed from the incubator and held at room temperature at least 30 minutes before proceeding with the VIP test. Alternatively, remove a small portion (e.g., 10 mL) from the enriched sample and hold at room temperature for 10 minutes before proceeding with the VIP test.

7.4.3 Transfer 0.1 mL of inoculated broth to sample addition well.

7.4.4 Incubate at ambient temperature for 10 minutes.

7.5 Reading Results:

7.5.1 Examine VIP unit for the presence of a distinct line in the test verification window. This line should be dark in colour when contrasted with the white background and extend across the window. Absence of a control line indicates an invalid test result. Contact BioControl Technical Services.

7.5.2 Observe test sample window. Presence of a distinct line, as described above, indicates a presumptive positive sample. Absence of a line is a negative. Differing intensities of test and control lines are acceptable as long as control line is present.

7.5.3 Positive and negative control cultures should be run concurrently with samples.

7.5.4 Autoclave units at 121°C for a minimum of 15 minutes prior to discarding.

7.6 Confirmation of Positive VIP Samples:

7.6.1 Presumptive positive samples must be confirmed culturally as described in MFLP-80. Concentrate presumptive positives using MFLP-90, or equivalent procedures, then proceed to specified selective agars and confirm biochemically and serologically.

8. REFERENCES

8.1 Warburton, Don. 2004. MFLP-80. Isolation of E. coli O157 in foods. In: Volume 3. Compendium of Methods. Website: http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php