MFLP-12: Identification of Escherichia coli O157:H7 and Verotoxin-producing Escherichia coli O157:NM by the ADIAFOOD Rapid Pathogen Detection System, a Real-time Polymerase Chain Reaction System

Laboratory Procedure MFLP-12
March 2009

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Identification of Escherichia coli O157:H7 and Verotoxin-producing Escherichia coli O157:NM Using the ADIAFOOD Rapid Pathogen Detection System, A Real-time PCR Technique

Stephen Shaw
Microbiologist, Canadian Food Inspection Agency
1400 Merivale Road,
Ottawa ON K1A 0Y9

E-mail: stephen.shaw@inspection.gc.ca

1. Application

The method is applicable to the rapid identification of Escherichia coli O157:H7 (E. coli O157:H7) strains and verotoxin-producing Escherichia coli O157:NM strains (E. coli O157:NM) isolated from raw ground beef using the standard E. coli O157:H7 culture technique, MFLP-80 (8.1), other HPFB methodologies, or the ADIAFOOD real-time PCR 8-hour enrichment methodology to detect E. coli O157:H7. It can be applied to the presumptive identification of E. coli O157:H7/NM from enrichment broth cultures. When product-based compliance action is anticipated, and where stipulated; HPFB methodology MFLP-80 (8.1) or equivalent is used for final confirmation of presumptive positive samples obtained from ADIAFOOD method.

This revised method replaces MFLP-12, dated January 2006.

2. Principle

Following the sample enrichment procedure for raw ground beef, the broth is subjected to a real-time PCR procedure in which the target pathogen DNA is amplified and detected using specific primers and molecular beacons. Molecular beacons consist of unique sequence probes that allow for the identification of the pathogen with a high level of specificity. Once bound to their target, the molecular beacons emit a fluorescent signal that is proportional to the amount of amplified pathogenic DNA. In the absence of target bacteria in food samples, no fluorescent signal is detected.

For the detection of E. coli O157:H7, two markers are used. The first detects the presence of E. coli O157 and the second confirms the presence of E. coli O157:H7. E. coli O157:NM (non-motile) strains that are verotoxin-producers, and thus pathogenic, will also be detected. The O157:NM strains that do not produce verotoxins will not. Taken together, positive results from these two markers will confirm the presence of E. coli O157:H7 or verotoxin-producing E. coli O157:NM contamination in the food sample.

The entire procedure, after the enrichment, identifies presumptive positive samples within 3 hours. This technique can replace the usual screening tests thus providing considerable savings on time, labour and cost of the analysis. The real-time PCR technique has proven to be a specific and sensitive method for the presumptive identification of E. coli O157:H7 and verotoxin-producing E. coli O157:NM in raw ground beef samples (8.2).

The ADIAFOOD Rapid Pathogen Detection System for E. coli O157:H7/NM has been AOAC-RI validated and was granted Performance Tested Method^SM status in 2004, Certificate No. 010409.

ADIAFOOD* is a registered trademark of AES Chemunex; (Rue Maryse Bastié - Ker Lann, CS 17219 - F-35172 Bruz Cedex, Phone: 33 (0)2 23 50 12 12, Fax: 33 (0)2 23 50 12 00, www.aes-lab.com). *Trademark is pending in Canada.

3. Definition of Terms

See Appendix A of Volume 3.

4. Collection of Samples

See Appendix B of Volume 3.

5. Specialized Molecular Markers, Reagents, Buffers, Materials and Equipment

Note: The laboratory Supervisor must ensure that completion of the analysis described in this method is done in accordance with the International Standards reference "ISO/IEC 17025:2005 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories".

5.1 Materials and Equipment

5.1.1 ADIAFOOD Kit components

Extraction buffer (EX-1)
Extraction reagent, lyophilized (EX-2)
Detection buffer (DT-1)
Detection reagent, lyophilized (DT-2)
Detection microplates or strip tubes containing pre-dispensed PCR reagents
Control strip tubes (only with Detection strip kits)
PCR optical-grade caps

Note: All kits are manufacturer by AES Chemunex Canada, www.aeschemunex.com and distributed in Canada by Innovation Diagnostics, www.innovationdiagnostics.com, 1-888-965-1871.

5.1.2 Additional equipment and consumables required

AES-validated Real-Time PCR thermocycler *
Conventional thermocycler for DNA extraction (optional)
Centrifuge with rotor and microplate carriers
Vortex equipped for microplates
Two multi-channel pipettors
Single-channel pipettor
PCR enclosure (optional)
PCR capping tool
Strip holders
ADIAFOOD Extraction kit (containing extraction plates, domed caps and seals)
Aerosol barrier tips for pipettors
Disposable multi-channel pipettor basins
Sterile microcentrifuge screw cap tubes (2 mL)
Powder-free gloves
Required enrichment media
Stomacher filter bags
Disposable serological pipettes
Alcohol (denatured) and bleach

*Real-time PCR thermocycler specifications: 96-well (or 48-well) microplate (low and high profile) or 96 (48) x 0.2 mL tubes (low and high profile) sample capacity and the ability to excite fluorophores with a peak excitation range of 485 to 520 nm and to detect fluorophores with a peak emission range of 500 to 600 nm.

5.2 Molecular Markers, Temperature Cycling Program, Buffers and Reagents

5.2.1 Molecular markers

The oligonucleotide primers and molecular beacon probes are supplied in the kit and their specificity for E. coli O157:H7/NM has been verified (8.2).

5.2.2 Temperature cycling programs

The temperature cycling programs for the PCR include an automated extraction cycle, resulting in the lysis of the bacterial cells, followed by an automated PCR detection process.

5.2.3 Buffers and reagents

All buffers and reagents are provided by AES Chemunex with each kit. These include the extraction reagents (EX-1, EX-2) and the detection reagents (DT-1, DT-2). All other PCR components are pre-dispensed in the wells of the detection microplate or strips. All water, pipettes, pipette tips and other materials coming in contact with samples or PCR reagents should be sterile and DNase free and/or autoclaved prior to use to remove any DNase and/or other contamination.

6. Procedure

6.1 Handling of Samples

Prepare and enrich samples according to the standard culture methodology for isolation of E. coli O157:H7 (8.1), other HPFB methodologies, or the ADIAFOOD 8-hour sample enrichment methodology, as per the manufacturer's instructions. Remove a portion of the culture enrichment and subject it to the PCR technique as indicated below. Positive and negative control wells are also processed with each sample batch.

6.2 DNA Extraction

6.2.1 Dispense the contents from EX-1 (squeeze bottle) into the EX-2 vial to obtain reconstituted EX-2.

6.2.2 Recap the EX-2 vial and mix by inversion to ensure complete dissolution of desiccated reagent.

6.2.3 Pour reconstituted EX-2 into a multi-channel pipettor basin.

6.2.4 Aliquot 90 μL of reconstituted EX-2 into each well of the Extraction microplate (3 wells per sample).

6.2.5 Transfer 10 μL of cell suspension from 6.1 into the Extraction microplate according to the sample assignment layout.

6.2.6 Seal the wells of the Extraction microplate with the domed cap strips provided.

6.2.7 Place the Extraction microplate into the thermocycler.

6.2.8 Begin the DNA extraction program on the thermocycler.

6.2.9 When the Extraction program has been completed, take out the Extraction microplate. All organisms are now lysed and inactivated.

6.2.10 Centrifuge the Extraction microplate for 5 minutes at 1,800 xg. The supernatant can now be used in the PCR amplification method.

6.3 PCR Amplification Method

6.3.1 Dispense the contents from DT-1 (squeeze bottle) into the DT-2 vial to obtain reconstituted DT-2.

6.3.2 Recap the DT-2 vial and mix by inversion to ensure complete dissolution of desiccated reagent.

6.3.3 Pour reconstituted DT-2 into a multi-channel pipettor basin.

6.3.4 Take out a Detection microplate or strips from its pouch (place them on the strip holder).

6.3.5 Aliquot 15 μL of reconstituted DT-2 into the wells of the Detection microplate or strips.

6.3.6 Transfer 10 μL of extracted DNA from the Extraction microplate into the wells of the Detection microplate or strips.

6.3.7 Seal the Detection microplate or strips with the optical-grade caps provided.

6.3.8 Seal the Extraction microplate using the provided microplate seals and store at 4°C until the analysis is completed.

6.3.9 Vigorously invert the microplate or strips 4 times to ensure that the contents are thoroughly mixed in the wells.

6.3.10 Gently tap the Detection microplate or strips to remove any air bubbles from the bottom of wells

6.3.11 Vortex for 1 minute at maximum speed.

6.3.12 Centrifuge the Detection microplate or strips for 1 minute at 1,800 x g to ensure that all the PCR mixture, including the sample's DNA extract, is in the bottom of the wells.

6.3.13 Secure the Detection microplate or strips tightly in the PCR instrument. Make sure well A1 is in the upper left corner. Click on "Start Detection" to initialize the PCR protocol.

6.3.14 When the Detection program has been completed, remove and discard the Detection microplate or strips.

7. Interpretation of Results

The amplicons (PCR products) generated from the E. coli O157/O157:H7/NM target sequences by this PCR method are double-stranded DNA fragments. A positive PCR test will result in a fluorescence response higher than the baseline produced by the negative controls within 40 cycles of the PCR amplification. The baseline cut-off fluorescence value is determined and set by the system. A negative PCR test will normally not produce visible fluorescence. If fluorescence occurs above the baseline cut-off value in the negative controls, the results of the test are invalidated and the analysis must be repeated with precautions taken to eliminate possible sources of error. Any sample found to be positive by the ADIAFOOD real-time PCR technique must be confirmed with the required biochemical tests prescribed in the HPFB, MFLP-80 (8.1) or equivalent methods.

8. References

8.1 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 website at: http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3/index-eng.php.

8.2 Warnex Research Inc. 2005. Validation of the Warnex Rapid Pathogen Detection System for Escherichia coli O157:H7/NM 8-Hour Enrichment in Raw Ground Beef (in-house unpublished data and AOAC data).