Detection Of E. Coli O157 By The Tecra® E. Coli O157 Visual Immunoassaytm (VIA) Method And Tecra E. Coli O157 Immunocapture

Laboratory Procedure MFLP-91
February 2006

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HEALTH PROTECTION BRANCH
OTTAWA

Don Warburton
Evaluation Division
Bureau of Microbial Hazards
Food Directorate, Health Canada
Postal locator: 2204A1
Ottawa, Ontario, K1A 0L2

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

1. Application

This method is applicable to the detection of E. coli O157 from foods, food ingredients and environmental samples to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. This revised method replaces MFLP-91, dated April 1997.

The TECRA E. coli O157 Visual Immunoassay is a rapid and specific screening system for the in vitro detection of E. coli O157 (including E. coli O157:H7 and other enterohaemorrhagic E.coli O157 strains) in food and environmental samples. After overnight enrichment the results can be obtained within 2 hours.

Presumptive positive results should be confirmed by using MFLP-80 (7.4). This is especially important in situations such as product recalls. Historically, E. coli O157 has been particularly difficult to confirm from enrichment cultures due to the problem of high background levels of competing organisms including other serotypes of E. coli. TECRA E. coli O157 Immunocapture (ICM) is designed to provide a convenient and rapid means of isolating E. coli O157 from enrichment cultures and may be used to facilitate isolation of E. coli O157 after obtaining a presumptive positive with the VIA.

Both the TECRA E.coli O157 VIA and ICM were given Performance Tested status by the Association of Official Analytical Chemists Research Institute (AOAC-RI, license number 001101).

2. Principle

The TECRA E. coli O157 Visual immunoassay is an Enzyme Linked Immunosorbent Assay (ELISA) performed in the "sandwich" configuration. High affinity "capture" antibodies specific for E. coli O157 have been adsorbed onto the surface of wells. If E. coli O157 antigens are present in the sample they are captured by the antibodies. All other material in the sample is washed away. The "sandwich" is completed by the addition of enzyme labelled antibodies (conjugate) specific for E. coli O157. The presence of E. coli O157 is indicated when the bound conjugate converts the substrate to a green color. Alternatively, if E. coli O157 is not present no green color develops. Total time to perform the test including overnight incubation is 20 hours.

If a presumptive positive E. coli O157 result is obtained with the VIA, a sample of the same enrichment broth may be added to Tube 1 of the ICM module together with the dipstick provided. Highly specific purified antibodies on the surface of the dipstick selectively capture any E. coli O157 present. After incubation in tube 1 and washing in tube 2, the dipstick is transferred to an enrichment broth in tube 3. During incubation, E. coli O157 is selectively captured on the dipstick and replicates to high levels. After incubation, tube 3 together with the dipstick, is vortexed to release the E. coli O157 bound to the dipstick. Dilutions are then spread-plated onto selective agar for confirmation.

TECRA® is the registered trademark of TECRA International Pty Ltd.13 Rodborough Rd, Frenchs Forest, NSW 2086 Australia.

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

5.1 Kit Components (VIA):

All reagents for the TECRA E. coli O157 Visual Immunoassay are provided in one kit, which contains enough material for a maximum of 94 tests (96 wells are provided but the positive and negative controls supplied with the kit must be run each time the test is performed). The kits are available in North America from Biotrace International Inc. at PO Box 746, Bothell, WA 98041 (mailing) or 21312 30^th Drive SE, Bothell WA 98021 (physical).  www.biotrace.com

Instructions
Sample record sheet
Plate holder
Resealing strip
Antibody coated microtitre wells
Reagent 1 - Wash concentrate
Reagent 2 - Positive control
Reagent 3 - Control diluent Reagent 4 - Conjugate
Reagent 5 - Conjugate diluent
Reagent 6 - Substrate
Reagent 7 - Substrate diluent
Reagent 8 - Stop Solution
Reagent 9 - Sample additive

5.2 Additional Requirements (VIA):

35-37°C incubator
41-43°C incubator
Serological pipettes
Plastic squeeze bottle
Pipette (20 µL, 50 µL and 200 µL)
Plastic wrap
Waterbath (100°C)
Small screw cap bottles or tubes capable of containing 5 mL and suitable for boiling.
Vortex mixer
Glass test tubes (15mm x 15cm)
Blender/stomacher/stomacher bags.

5.3 Media (VIA)

Media are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 for the formula of individual media.

Enrichment broth media:
Modified EC medium with Novobiocin (mEC + N). TECRA product MECMED500.
Buffered Tryptone Soy Broth with Novobiocin (BTSB + N).

5.4 Kit Components (ICM)

All reagents for the TECRA E. coli O157 Immunocapture are provided in one kit containing 20 test modules. The kits are available in North America from Biotrace International Inc. at PO Box 746, Bothell, WA 98041 (mailing) or 21312 30th Drive SE ,Bothell WA 98021 (physical).  www.biotrace.com

Instructions
20 x antibody-coated dipsticks
20 x 3 tube Immunocapture modules containing:
Reagent 1 -Buffer
Reagent 2 -Wash solution
Reagent 3 -Tryptone soya broth

5.5 Additional Requirements (ICM):

Pipettes, 4mL
Incubators, 35-37°C and 41-43°C
Vortex mixer

5.6 Media (ICM):

Media are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 for the formula of individual media.

Sterile diluent (4.5mL) in tubes eg. Saline or 0.1% peptone
CT-SMAC plates
At least one additional agar from MFLP-80.

6. Procedure

6.1 Handling of Sample Units

6.1.1 Analyze samples as soon as possible. If necessary, store samples under time and emperature conditions that will prevent the growth or death of native microflora. If sample units have been abused in transit, resampling of the lot should be carried out.

1. Frozen Foods: Sample units that show no signs of thawing upon receipt may be stored in the freezer at -10ºC to -20ºC.
2. Dried and shelf stable foods may be stored at room temperature (20 to 25ºC).
3. Refrigerate all other foods (2 to 8ºC, including those that are received in a partially thawed condition). Analyze these samples as soon as possible preferably within 24 h of receipt.

6.1.2 Thaw frozen samples at room temperature within 60 min; if this is not possible, thaw the samples at refrigerator temperature (2 to 8ºC).

6.1.3 If the sample unit received for analysis is less than the recommended analytical unit, analyze the entire amount and record the weight used.

6.1.4 Blending of samples should be limited to the minimum time required to produce a homogeneous suspension. Excessive blending could result in physical damage that would adversely affect the viability of endogenous microflora.

For products that do not require blending, disperse the analytical unit into the appropriate pre-enrichment broth.

6.1.5 Use aseptic techniques and sterile equipment at all stages of analysis. Containment during the handling of powdered products is critical if cross-contamination of the work environment is to be avoided.

6.2 Enrichment

Samples should be enriched according to the appropriate enrichment protocol in the TECRA E. coli O157 Visual Immunoassay Methods Manual. Enrichment broths must be brought to room temperature (20-25°C) before combining with samples. When testing volumes greater than 225 mL, enrichment broths should be prewarmed before addition of samples.

6.3 Analysis

After overnight incubation samples should be heat killed and tested in the VIA kit in accordance with the TECRA E. coli O157 Visual Immunoassay Instruction Manual. The immunoassay will take approximately 2 hours and may be read visually or with the aid of a spectrophotometer.

6.4 Cultural Confirmation

Samples positive by the TECRA E. coli O157 Visual Immunoassay should be confirmed from the enrichment broth by using the TECRA E. coli O157 Immunocapture according to the ICM Instruction Manual. Immunoenrichment in the ICM module will take approximately 4 hours during which time the numbers of E. coli O157 cells will increase compared to competitive flora. The broth from tube 3 of the module is then spread plated onto CT-SMAC agar and one additional agar from MFLP-80. Typical colonies are confirmed following MFLP-80.

7. References

7.1 AOAC Research Institute Independent Laboratory Study Report. AOAC-RI license number 001101. Stephen Shaw. 1999. Ottawa Laboratory Carling, Canadian Food Inspection Agency, Ottawa, Canada.

7.2 International Commission on Microbiological Specifications for Foods (ICMSF).1986. Microorganisms in Foods 2. Sampling for microbiological analysis: Principles and Specific applications. Second edition. University of Toronto Press, Toronto, ON.

7.3 Feng, P. and Weagant, S.D. US Food and Drug Administration. Bacteriological Analytical Manual Online. 8th Edition, Revision A, 1998. Chapter 4A- Diarrheagenic Escherichia coli. Available at http://www.cfsan.fda.gov/~ebam/bam-4a.html.

7.4 Warburton, D. and D. Christensen. 2006. MFLP-80. Isolation of E. coli O157:H7 or NM in Foods. In: Volume 3. Compendium of Analytical Methods. Available at the Health Canada (HC) website at http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3/index-eng.php.