Identification of Listeria Monocytogenes by the Warnex™ Real Time Polymerase Chain Reaction System

Laboratory Procedure MFLP-31
May 2005

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Health Products and Food Branch
Ottawa

Stephen Shaw and Jessica Bosley
Technology Development Section, Ottawa Laboratory Carling (OLC) Canadian Food Inspection Agency,
Ottawa, Ontario, K1A 0C6

E-mail: sshaw@inspection.gc.ca
bosleyj@inspection.gc.ca

1. APPLICATION

The method is applicable to the rapid identification of Listeria monocytogenes strains (LM) isolated from foods and other samples using the standard Listeria culture techniques, MFHPB-30 (6.1), MFLP-74 (6.2), other HPFB methodologies and other non-compendium methodologies to detect Listeria species. It can be applied to the presumptive identification of Listeria species from selective enrichment broth cultures. When product-based compliance action is anticipated, and where stipulated, HPFB methodology should be used exclusively or for further confirmation of real time polymerase chain reaction (PCR) positive colonies.

This revised method replaces MFLP-31, dated June 2003.

2. PRINCIPLE

Following the selective enrichment procedure, (Fraser broth, Fbr) for food, food ingredients and environmental samples, the broth is subjected to a real time PCR procedure which amplifies a unique specific fragment of DNA sequence to the LM gene. The oligonucleotide primers used in the fluorescent PCR system are highly specific for LM, and do not amplify DNA present in other non-LM strains under the reaction conditions. The resulting amplified DNA fragment has a specific molecular size, defined by the selected primers, and is readily identified by the LM specific fluorescent probe. The entire procedure, after the selective enrichment step (Fbr), 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 LM strains from a variety of samples.

The Warnex™ real time PCR rapid pathogen detection system for Listeria monocytogenes has been AOAC-RI validated and was granted "Performance Tested Method" status in 2004, Certificate No. 040402.

* Warnex™ is a trademark of  Warnex Diagnostics Inc., 3885 Industriel Blvd., Laval (Quebec), Canada, H7L 4S3.
Ph: (450) 663-6724, Fax: (450) 669-2784, website: http://www.warnex.ca

3. SPECIALIZED PRIMERS, REAGENTS, BUFFERS, MATERIALS AND EQUIPMENT

3.1. MATERIALS AND EQUIPMENT

3.1.1 Materials and special equipment provided

Extraction buffer (EX-1)
Extraction reagent, lyophilized (EX-2)
Extraction plates
Plate seals
Detection buffer (DT-1)
Detection reagent, lyophilized (DT-2)
Detection plates (containing pre-dispensed PCR reagents and primers)
PCR optical-grade plastic caps

3.1.2 Additional materials and special equipment required

Warnex-validated Real-Time PCR thermocycler *

* Thermocycler specifications:
- 96 well low profile microplate or 96 x 0.2 mL low profile strip tube sample capacity.
- ability to excite fluorophores with a peak excitation range of 485 to 520 nm
- ability to detect fluorophores with a peak emission range of 500 to 600 nm.

Vortex (with platform head adaptors and replacement inserts)
Centrifuge (with rotor and plate adaptor)
Stomacher
Stomacher filter bags
Pipettors to cover range of volumes (0.5-1000 μL) with sterile plugged pipette tips.

3.2 PCR PRIMERS, TEMPERATURE CYCLING PROGRAM, BUFFERS AND REAGENTS

3.2.1 PCR Primers

The oligonucleotide primers and molecular beacon probes are supplied in the kit and their specificity for LM has been verified (6.3).

3.2.2 Temperature cycling programs

The temperature cycling programs for the PCR include an automated 15-minute extraction cycle, resulting in the lysis of the bacterial cells, followed by an automated PCR detection process. The thermal cycler program for the detection (amplification) process is preset for the following sequence of cycling parameters:

Amplification:

1 cycle of:
Hot start, 15 mins, 95°C

40 cycles of:
Denaturation, 15 secs, 94°C
Annealing, 15 secs, 55°C
Plate read
Extension, 15 secs, 72°C

3.2.3 Buffers and Reagents

All buffers and reagents are provided by Warnex Diagnostics Inc. with each kit. These include the extraction reagents (EX-1, EX-2) and the detection reagents (DT-1, DT-2). All other detection buffers and reagents are pre-dispensed in the detection plate wells. 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.

4. PROCEDURE

4.1 Handling of samples

Sample units are handled and subjected to the enrichment, isolation and plating procedures according to Health Canada Listeria methodology (6.1, 6.2) or other validated Listeria methodology. The Fbr broth is then sampled and subjected to the PCR technique as indicated below. A positive control consisting of a broth culture of LM strain is also processed along with each set of samples as well as a negative control consisting of non-LM organism. A negative reagent/sterility control is also processed with each sample set.

4.2 DNA Extraction

4.2.1 From the kit, dispense the contents from EX-1 (squeeze bottle) into the EX-2 vial to obtain reconstituted EX-2. Recap the EX-2 vial and mix by inversion to ensure complete dissolution of desiccated reagent. Pour reconstituted EX-2 into a multi-channel pipettor basin.

4.2.2 Aliquot 90 μL of reconstituted EX-2 into each well of a 96-well Extraction plate (one well per extraction).

4.2.3 Transfer 10 μL of cell suspension from 4.1 into the Extraction plate according to the predetermined plate layout using a single channel pipettor.

4.2.4 Seal the wells of the Extraction plate with the optical-grade plate cap strips provided.

4.2.5 Place the Extraction plate into the thermocycler and close the lid.

4.2.6 Begin the DNA extraction program for the thermocycler.

4.2.7 When the Extraction program has been completed, take out the Extraction plate.

4.2.8 All organisms are now lysed and inactivated.

4.2.9 Centrifuge the Extraction plate for 5 minutes at 1,800 x g.

4.2.10 The supernatant can now be used in the PCR amplification method.

4.3 PCR Amplification method

4.3.1 From the kit, dispense the contents from DT-1 (squeeze bottle) into the DT-2 vial to obtain reconstituted DT-2. Recap the DT-2 vial and mix by inversion to ensure complete dissolution of desiccated reagent. Pour reconstituted DT-2 into a multi-channel pipettor basin.

4.3.2 From the kit, take out a Detection plate and remove the pouch.

4.3.3 Aliquot 15 μL of reconstituted DT-2 into the 96-well Detection plate using a multi-channel pipettor.

4.3.4 Transfer 10 μL of extracted DNA from the Extraction plate into the wells of the Detection plate.

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

4.3.6 Seal the Detection plate with the optical-grade plate cap strips provided.

4.3.7 Vortex the Detection plate for 1 minute.

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

4.3.9 Secure the Detection plate tightly in the PCR instrument. Make sure well A1 is in the upper left corner. Close the PCR lid, click on "Start Detection" to initialize the PCR protocol.

4.3.10 When the Detection program has been completed, remove and discard the Detection plate.

5. INTERPRETATION OF RESULTS

The amplicon (PCR product) generated from the Listeria monocytogenes gene sequence by this PCR method is a double stranded DNA fragment. 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 PCR apparatus. 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 Warnex PCR technique must be confirmed culturally, especially when compliance action is anticipated.

6. REFERENCES

6.1 Pagotto, F., E. Daley, J. Farber and D. Warburton. 2001. MFHPB-30. Isolation of Listeria monocytogenes from all Foods and Environmental Samples. In: Volume 2, Compendium of Analytical Methods. Website:
http://www.hc-sc.gc.ca/fn-an/index-eng.php.

6.2 Pagotto, F., E. Daley and J. Farber. 2002. MFLP-74. Enumeration of Listeria monocytogenes in Foods. In: Volume 3, Compendium of Analytical Methods. Website: http://www.hc-sc.gc.ca/fn-an/index-eng.php.

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