Detection Of Escherichia coli O157:H7 In Foods - Assurance GDS™ For E. coli O157:H7 Gene Detection System

Laboratory Procedure MFLP-16
August 2005

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Health Products and Food Branch
Ottawa

Microbiological Methods Committee
Evaluation Division,
Bureau of Microbial Hazards,
Food Directorate,
Health Products and Food Branch,
Sir Frederick G. Banting Research Centre [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:H7 and toxigenic E. coli O157:NM (non-motile) in raw ground beef, beef trim, orange juice, apple juice, fresh vegetables and sprout process water to determine compliance with the requirements of Section 4 and 7 of the Food and Drugs Act.

2. Description

The Assurance GDS for E. coli O157:H7 (Assurance GDS) is a gene-based assay for the detection of E. coli O157:H7 in selected foods. This method is designed to be highly selective, and does not detect microorganisms that are potential cross reactors in antibody-based assays, including E. coli O157 that are not H7 or NM, and other microorganisms that express O157 antigen but are not E. coli O157:H7.

The method utilizes a 6.5 hour enrichment for raw ground beef, beef trim, orange juice, apple juice, fresh vegetables and an 8 hour enrichment for sprout process water. This is followed by a short sample preparation step, and automated amplification and detection process of 75 mins in a rotary instrument. For most foods, total time from starting sample enrichment to final results read out is approximately 8 hours.

3. Principle

The Assurance GDS system incorporates a sample concentration step, proprietary genetic detection reagents, and a multi-channel, rotary instrument platform to achieve fast and accurate results. After enrichment, populations of target microorganisms are concentrated using a proprietary concentration device and reagents or an automated sample concentration instrument. The step provides two benefits. First, the technique concentrates cell populations. Second, it physically segregates target microorganisms from both competitive microflora and the food matrix. This also affords easier identification of suspect colonies on agar plates.

Each concentrated samples is then transferred to a reaction tube containing amplification reagents. The reagent system utilizes specific primers and proprietary probes directed against a highly conserved DNA sequence in the target organism. The reaction tube is then sealed and placed in a proprietary multi-channel instrument which allows for simultaneous amplification and detection by on-line optical analysis. If the target sequence is present, a specific fluorescence will be generated and read automatically. A procedural control is also contained in every reaction tube and a different distinctive fluorescence is read simultaneously and separately by the instrument. All sample determinations, positive and negative, are indicated at the end of analysis.

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. Assurance GDS for E. coli O157:H7 kit - Catalog no. 61007-100 sufficient for 100 tests.*  BioControl Systems http://www.biocontrolsys.com, 800-245-0113) consisting of:
   
   Assurance GDS Reaction tubes containing lyophilized reagents needed for E. coli O157:H7 genetic detection and an internal positive control, 25 strips of 4 reaction tubes.
   Assurance GDS Sample Concentration Reagent, 1 vial.
   Assurance GDS Resuspension Buffer, 1 vial.
   Assurance Polymerase Dilution Buffer, 1 vial.
   
   
2. Hot start polymerase enzyme. ( http://www.eppendorf.com) or other suitable source. To be added to Assurance Polymerase dilution buffer from kit. Invert the tube 5 times to mix. Record the date on the tube. Prepared polymerase buffer solution can be stored up to 1 month at 2-8°C.
   
   
3. Enrichment broth
   BioControl mEHEC media (Catalog number 65012)
   
   
4. Materials (provided with start-up package)
   
   Equipment
   Assurance GDS Rotor-Gene system, computer and software.
   Assurance GDS sample concentration device (PickPen™ or equivalent).
   Assurance GDS vortex mixer
   Various calibrated pipets
   Aluminum block.
   
   Supplies
   Sample blocks
   Resuspension plates
   Tips for pipettes and PickPen
   Adhesive film

7. Procedure

7.1 Sample Enrichment

7.1.1. Prepare mEHEC media. For 25g sample, prewarm 225 mL sterile deionized water at 42°C overnight. On day of use aseptically transfer 7.1 g of BioControl mEHEC™ media into the prewarmed sterile water. Mix to dissolve the powder. Use prepared medium within 6 h. Alternatively, media can be prepared in advance and autoclaved.

7.1.2. Aseptically weigh 25 g sample into 225 mL prewarmed (42°C) mEHEC. Masticate sample for 2 min. If larger sample sizes are analyzed, proportionately increase the volume of mEHEC to maintain a 1:9 dilution ratio. Incubate for 6.5-18 h at 42°C.

7.2 Performing the Assurance GDS for E. coli O157:H7 Assay

7.2.1. Vortex concentration reagent. Transfer 20 μL to each of the required number of wells on the sample block.

7.2.2. Add 1 mL incubated sample to each sample block well containing concentration reagent. Immediately return samples to 42°C incubator. Seal sample block with adhesive film and place on the vortex shaker for 5 min at 600 rpm.

7.2.3. Remove and discard sealing film after vortex mixing is complete. Add 35 μL of resuspension buffer to the required number of wells in the resuspension plate

7.2.4. Load tips onto the PickPen. Extend the magnetic tips of the PickPen and insert into the first row of the sample block. Stir gently for 30 seconds while continually moving up and down from the surface to the bottom of the well.

7.2.5. Transfer PickPen to the corresponding row of the prepared resuspension plate. With tips submerged, retract the magnetic tips and tap gently to release particles into the resuspension buffer. Repeat for all wells on sample block using new tips for each row of samples.

7.2.6. Cool aluminum block at 2-8°C for at least 20 min prior to use. Place the required number of Assurance GDS reaction tubes in the aluminum block.

7.2.7. Open caps of reaction tubes in the aluminum block. Transfer 10 μL prepared polymerase buffer solution to each reaction tube.

7.2.8. Transfer 20 μL from each resuspension plate well into each reaction tube. Firmly press down on each reaction tube lid to close.

7.2.9. Place reaction tubes into Assurance Rotor-Gene in sequential order beginning with position #1 and run program.

7.3. Assay Results

7.3.1. Upon completion of the run, the Assurance Rotor-Gene program will provide a results table. Each sample that is "Positive" by the Assurance GDS must be culturally confirmed by the method MFLP-80 (8.1). "Negative" indicating that the sample is negative for E. coli O157:H7, or "No Amp" indicating that amplification did not occur. GDS positive samples must be confirmed culturally as outlined in MFLP-80 (8.1) . For "No Amp" results, contact BioControl Systems Technical Service 800245 0113

7.3.2. When the assay is completed, handle and dispose of all waste as a biohazard. Never open reaction tubes after amplification has started under any circumstances.

8. References

8.1 Warburton, Don. 2001. MFLP-80. Isolation of E. coli O157 in foods. In: Volume 3. Compendium of Methods. Webite:
http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3/index-eng.php