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Microbiological Methods Committee
Microbiology Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, Health Canada
Postal Locator: 2204E
Ottawa, Ontario K1A 0K9
This method is applicable to the rapid detection of Escherichia coli O157:H7 to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. Positive results must be confirmed with a cultural method. This method has been validated for use in raw processed meats, raw unprocessed meats and leafy greens.
Note: While this method is only approved for certain food products, as listed above, it is assumed that this method could be used with other foods. To ensure the method is fit for purpose for commodities outside the application, it is imperative that other commodities be properly validated following the criteria in the Compendium of Analytical Methods. It is requested that these validation data be forwarded to the Microbiological Methods Committee so the Application Section can be expanded to include these new foods if the data fulfil MMC requirements (refer to Development of Methods in Volume 1 of the Compendium of Analytical Methods).
The VIDAS« UP E. coli O157 (including H7) method, also known as the E. coli Phage Technology (ECPT) method, is an automated assay based on the ELFA technique (Enzyme-Linked Fluorescent Assay) for use on the VIDAS« family of instruments. The Solid Phase Receptacle (SPR«), serves as the solid phase as well as the pipetting device. The interior of the SPR« is coated with recombinant phage tail fiber protein for the capture of E. coli O157 including H7. Reagents for the assay are ready-to-use and pre-dispensed in the sealed reagent strips.
All of the assay steps are performed automatically by the VIDAS« instrument. The reaction medium is cycled in and out of the SPR« several times. Part of the enrichment broth is dispensed into the reagent strip. The E. coli O157 including H7 present are captured by the recombinant phage protein coating the interior of the SPR«. Unbound components are eliminated during the washing steps. Alkaline phosphatase conjugate is then cycled in and out of the SPR« and will bind to any E. coli O157 including H7 which are themselves bound to the phage protein on the SPR« wall. A final wash step removes unbound conjugate.
During the final detection step, the substrate (4-Methylumbelliferyl phosphate) is cycled in and out of the SPR«. The conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4-Methylumbelliferone) the fluorescence of which is measured at 450 nm. At the end of the assay, the results are analyzed automatically by the instrument. A test value, which is compared to stored standards (thresholds) and an interpretation (positive, negative) are generated for each sample.
See Appendix A of Volume 3.
See Appendix B of Volume 3.
Note: The Laboratory Supervisor must ensure that the analysis described in this method is carried out in accordance with International Standard referred to as "ISO/IEC 17025:2005 or latest version: General requirements for the Competence of testing and Calibration Laboratories" (7.1).
Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.
Mandatory media and instruments (from bioMÚrieux Canada, Inc, 1-800-361-7321)
1) VIDAS« family instrument (mini-VIDAS or VIDAS 30)
2) VIDAS« UP E. coli O157 kit (ref. 30122): contains the necessary reagents including standards and controls for the analysis of 30 test samples.
3) VIDAS« Heat and Go, or water bath (95-100°C) or equivalent system
Other materials and instruments
The media listed below are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 1 for the formulae of individual media.
4) Buffered Peptone Water (BPW)
5) Stomacher and stomacher bags, blender or equivalent
6) Pipette calibrated to dispense 500 μl and disposable tips
7) Incubators capable of maintaining 41.5°C
Note: It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures. Where 35°C is recommended in the text of the method, the incubator may be at 35 ▒1.0°C. Similarly, lower temperatures of 30 or 25 may be ▒ 1.0°C. However, where higher temperatures are recommended, such as 43 or 44.5°C, it is imperative that the incubators be maintained within 0.5°C and water baths be maintained within 0.2°C due to potential lethality of higher temperatures on the microorganism(s) being isolated.
The test shall be carried out in accordance with the following instructions:
6.1.1 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.
6.1.2 Analyze the sample units as soon as possible after receipt at the laboratory
6.2.1 Have sterile Buffered Peptone Water available, pre-warmed to 41.5 ▒ 0.5°C.
6.2.2 Clean the surface of the working area with a suitable disinfectant.
Combine portions from several locations within each solid sample unit, to ensure a representative analytical unit.
6.4.1 Aseptically place 25 g of sample in a Stomacher-type bag.
6.4.2 Add 225 ml of Buffered Peptone Water.
6.4.3 Mix for 2 minutes using a Stomacher-type mixer.
6.4.4 Incubate for 7-24 hours at 41.5 ▒ 0.5°C.
6.5.1 Aseptically place X g of sample in a Stomacher-type bag.
6.5.2 Add 3X ml of Buffered Peptone Water + vancomycin (8 mg/l).
6.5.3 Mix for 2 minutes using a Stomacher-type mixer.
6.5.4 For samples up to 75 g, incubate for 8-24 hours at 41.5 ▒ 0.5°C. For samples larger than 75 g, incubate for 10-24 hours at 41.5 ▒ 0.5°C.
6.6.1 Aseptically place 25 g of sample in a Stomacher-type bag.
6.6.2 Add 225 ml of Buffered Peptone Water + vancomycin (8 mg/l).
6.6.3 Mix for 2 minutes using a Stomacher-type mixer.
6.6.4 Incubate for 8-24 hours at 41.5°C ▒ 0.5°C.
6.7.1 Remove the VIDAS« UP E. coli O157 (including H7) kit from the refrigerator. Only remove the required reagents needed and allow them to come to room temperature for at least 30 minutes. Use one strip and one SPR« for each sample, control or standard to be tested. Make sure the storage pouch has been carefully resealed after the required SPR«s have been removed.
Note: Components of different lots cannot be mixed and cannot be used past the expiry date.
Note: When a new kit lot is being used, specifications (i.e., the factory master calibration curve data) must first be entered into the system using the master lot entry card included with each kit. Calibration, using the standard provided in the kit, must be performed for each lot of reagents upon opening, and every 14 days thereafter. Please refer to the package insert for the VIDAS« PTC protocol data entry, master lot data entry and calibration instructions.
6.7.2 After incubation, homogenize the enrichment broth.
6.7.3 Procedure for 25g of raw meat (processed or unprocessed)
184.108.40.206 Prepare the sample for the VIDAS assay by either using a water bath (or equivalent) or the VIDAS Heat and Go Device.
220.127.116.11 If the VIDAS« Heat and Go is used, transfer 0.5 ml of the enrichment broth into the sample well on the strip. Heat for 5 ▒ 1 minutes (see the VIDAS« Heat and Go Operator's Manual). Remove the strip and leave to cool for 10 minutes.
18.104.22.168 If a water-bath is used, transfer 1-2 ml of the enrichment broth into a tube. Seal the tube. Heat for 5 ▒ 1 minutes at 95-100°C. Cool the tube (do not mix). Transfer 0.5 ml of boiled broth into the sample well on the VIDAS« strip.
22.214.171.124 Proceed to step 6.7.6.
6.7.4 Procedure for 65 g to 375 g of raw meat (processed or unprocessed)
126.96.36.199 Transfer 2-3 ml of the enrichment broth into a tube. Seal the tube. Heat in a water-bath (or equivalent) for 5 ▒ 1 minutes at 95-100°C. Cool the tube (do not mix). Transfer 0.5 ml of boiled broth into the sample well on the VIDAS« strip.
188.8.131.52 Proceed to step 6.7.6.
6.7.5 Procedure for Leafy Greens
184.108.40.206 Prepare the sample for the VIDAS assay by either using a water bath (or equivalent) or the VIDAS Heat and Go Device.
220.127.116.11 If the VIDAS« Heat and Go is used, transfer 0.5 ml of the enrichment broth into the sample well on the strip. Heat for 5 ▒ 1 minutes (see the VIDAS« Heat and Go Operator's Manual). Remove the strip and leave to cool for 10 minutes.
18.104.22.168 If a water-bath is used, transfer 1-2 ml of the enrichment broth into a tube. Seal the tube. Heat for 5 ▒ 1 minutes at 95-100°C. Cool the tube. Mix the boiled broth and transfer 0.5 ml into the sample well on the VIDAS« strip.
22.214.171.124 Proceed to step 6.7.6.
Note: Do not heat the standard and controls
6.7.6 Insert the SPR« and the strip into the instrument. Check to make sure the colour labels with the assay code on the SPR« and the reagent strips match.
6.7.7 Initiate the assay as directed in the Operator's manual.
6.7.8 After the assay is completed, remove the SPR« and the strips from the instrument. Dispose the used SPR«'s and strips into an appropriate biohazard receptacle in accordance with applicable local regulations.
Once the assay is completed, the computer analyzes results automatically. A result with a Test Value greater than or equal to 0.04 indicates the presumptive presence of E. coli O157 in the test sample.
Using the unheated enrichment broth, proceed with the plating and confirmation steps of a cultural method (i.e., MFLP-80). .
7.1 ISO/IEC 17025:2005. General requirements for the competence of testing and calibration laboratories.
7.2 Warburton, D., and Christensen, D. 2008. Isolation of E. coli O157:H7 or NM in foods (MFLP-80). Volume 2. The Compendium of Analytical Methods. http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php
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