MFLP-32 Identification of Salmonella species using the Adiafood rapid pathogen detection system, a real-time pcr technique

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Laboratory Procedure MFLP-32
May 2011

Health Products and Food Branch
Ottawa

Microbiological Methods Committee
Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Postal Locator: 2204E
HPFB, Ottawa, Ontario, K1A 0K9

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

1. Application

The method is applicable to the rapid identification of Salmonella species isolated from foods and other samples using the standard Salmonella culture techniques, MFHPB-20 (8.1), MFLP-75 (8.2), other HPFB methodologies and other non-compendium methodologies to detect Salmonella species. It can be applied to the presumptive identification of Salmonella species from enrichment broth cultures. When product-based compliance action is anticipated, and where stipulated; HPFB methodology, MFHPB-20 (8.1), MFLP-75 (8.2) or equivalent is used for final confirmation of presumptive positive samples obtained from ADIAFOOD method.

This revised method replaces MFLP-32, dated January 2010.

2. Principle

Following the sample enrichment procedures, pre-enrichment (PE) for animal feeds, fertilizers and environmental samples or selective enrichment (SE) for food and food ingredients, the broths are subjected to a real time PCR 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 since the oligonucleotide primers used within this system are highly specific for Salmonella spp, and do not amplify DNA present in other non-Salmonella organisms under the same reaction conditions.

The entire procedure, after the enrichment steps (PE, SE), identifies presumptive positive samples within 3 hours, and 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 Salmonella species from a variety of samples (8.3).

The ADIAFOOD Rapid Pathogen Detection System for Salmonella species has been AOAC-RI validated and was granted "Performance Tested Method" status in 2004, Certificate No. 070402.

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 17025:1999 (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) × 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 Salmonella has been verified (8.3).

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 Salmonella species (6.1, 6.2) or other validated Salmonella methodology. 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 (2 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 × g. 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 × 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 Salmonella gene sequence 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 culturally, especially when compliance action is anticipated.

8. References

8.1 D’Aoust, J-Y, and U. Purvis. 1998. MFHPB-20. Isolation and Identification of Salmonella from Foods. In: Volume 2, Compendium of Analytical Methods.

8.2 Poppe, C., E. D. Mann, D. W. Warburton and S. J. Shaw. 2004. MFLP-75. Procedure for the Isolation of Salmonella species by the Modified Semi-Solid Rappaport-Vassiliadis (MSRV). In: Volume 3, Compendium of Analytical Methods.

8.3 Warnex Research Inc. 2002. Validation of the Salmonella Real Time PCR Technology for the Rapid Identification of Salmonella species from Food and other Samples. Personal Communication (unpublished data).

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