The detection of Listeria species from environmental surfaces using the Dupont Qualicon BAX® System method and direct plating

Laboratory Procedure MFLP-15
February 2011

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Don Warburton and Franco Pagotto
Research Division, Bureau of Microbial Hazards,
Food Directorate, Health Canada, Ottawa, Ontario, HPFB, Postal Locator 2204E
K1A 0K9

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

1. Application

This method is applicable to the rapid detection of Listeria species to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. This method has been validated for use on environmental samples. This method can be used to detect Listeria spp. and/or rapidly detect Listeria monocytogenes by plating the BAX® system media on selective agars as well as analyzing aliquots of this broth via the BAX® system instrument. This revised method replaces MFLP-15, dated February 2009.

2. Description

The BAX® system is a convenient yes/no screening tool that uses the Polymerase Chain Reaction (PCR) technology for rapid amplification and fluorescent detection. Food processors and associated laboratories can use the BAX® system as a quick method for accurately detecting Listeria species from a wide variety of surfaces. Following enrichment, sample preparation involves about 1.5 hours of user time, and the automated procedure delivers reliable results about 4 hours later. The BAX® system is designed for use by qualified lab personnel who follow standard microbiology practices. The original BAX® system screening protocol has been validated against the USDA-FSIS culture method (8.2) for detecting Listeria spp. in environmental samples. This modified method was validated against MFHPB-30 (Warburton 2008, unpublished data).

3. Principle

The BAX® system uses the Polymerase Chain Reaction (PCR) to amplify a specific fragment of bacterial DNA, which is stable and unaffected by growth environment. The fragment is a genetic sequence that is unique to Listeria spp., thus providing a highly reliable indicator that the organism is present. The automated BAX® system then uses fluorescent detection to analyze PCR product for positive or negative results.

PCR is a powerful means for quickly providing millions of copies of a specific DNA fragment. In a typical application, sample DNA is combined with polymerase, nucleotides and primers that are specific for a given nucleotide sequence. This mixture then undergoes a series of timed heating and cooling cycles.

The BAX® system simplifies this process by combining the primers, polymerase, nucleotides and positive control into a single sample tablet that is already packaged inside the PCR tubes. Additionally, the automated fluorescent detection allows for closed-tube testing, minimizing the potential for carry-over contamination with amplified DNA.

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

6.1. BAX® System PCR Assay for Screening Genus Listeria - (Cat. No. D11000147,; sufficient for 96 tests - DuPont Qualicon, phone: 1-800-863-6842, fax: 302-695-5301). PCR tablets, packaged 1 tablet per PCR tube, in 12 strips of 8 tubes. The tablets include the reagents needed for the test reaction as well as an internal positive control (which eliminates the need to run a separate QC reaction). Tablets are 7.6 ± 0.1 mg. Optical caps, 12 strips of 8 caps. Lysis buffer, 12 mL/bottle, pH 8.35 ± 0.05 at 25°C. Used to prepare working lysis reagent. Protease solution, 400 µL/vial. Used to prepare working lysis reagent.

6.2. BAX® System Enrichment Media for Listeria (Cat. No. D12232794).
Follow manufacturer's instructions.

Note: This is the only enrichment broth presently validated for use in this method.

6.3. D/E Neutralizing broth - Difco product number 281910 (Fisher Scientific # DF0819-17-2) or equivalent.

6.4. Whirl-Pak bags and sponges - 18 oz Whirl-Pak® "Speci-Sponge®" Environmental Surface Sampling Bag (Nasco product number B01245WA or equivalent).

6.5. Cotton tipped swabs - Puritan product number 867-WCNOGLUE or equivalent.

6.6. Materials (included with BAX® system start-up package).

6.6.1. Cycler/detector with verification plates.
6.6.2. Computer workstation with Microsoft Windows ® operating system, BAX® system application, and printer.
6.6.3. Dry block heaters with thermometers and inserts for lysis tubes.
6.6.4. Capping/decapping tools.
6.6.5. Pipettes for reagent and sample transfers (5 μl, 10μl, 10-50 μl 8 channel, repeat pipettor).
6.6.6. Cooling block with inserts for lysis tubes and PCR tubes.
6.6.7. PCR tube holders.
6.6.8. Lysis tubes with caps and rack.
6.6.9. Tips for pipettes.
6.6.10. Powder-free nitrile gloves.
6.6.11. BAX® system User Guide.

6.7. Additional materials

6.7.1. Incubator capable of maintaining a temperature of 35 ± 1°C.
6.7.2. Microcentrifuge.
6.7.3. Microfuge tubes (1.5 mL).
6.7.4 Vortex
6.7.5. Molecular grade water.
6.7.6 Plating and confirmation media and supplies from MFHPB-30.
Note: chromogenic agars are needed only for detecting L. monocytogenes

7. Procedure

7.1. Collect and Enrich Samples

7.1.1. Pre-warm prepared BAX® system Listeria enrichment broth at 35 ± 1°C prior to testing.

7.1.2. Surface samples collected by sponge; Moisten sponges with 10 mL of D/E Neutralizing Broth and store within whirl-pak bags.

7.1.2.1. A minimum of 2 hours after plant/equipment sanitation, obtain a sample by swabbing a 10 cm x 10 cm (100 cm²) section of the surface with a sponge.

7.1.2.2. After returning the sponge to the sample bag, add 190 mL of BAX® system media for Listeria genus. Homogenize by hand for 1-2 min.

7.1.2.3. Incubate for 48 ± 2 h at 35 ± 1°C. If screening the enrichment broth with the BAX® system proceed with step 7.1.4 otherwise follow the isolation procedure of MFHPB-30 (see step 7.6).

7.1.3. Surface samples collected by swab. Pre-moisten cotton swabs with 0.5 mL of neutralizing broth (D/E Neutralizing broth).

7.1.3.1. A minimum of 2 hours after plant/equipment sanitation, obtain a sample by swabbing a 2.5 cm x 2.5 cm (6.25 cm²) section of the surface with the swab.

7.1.3.2. Return swabs to 10 mL of BAX® system media for Listeria genus and rotate by hand for 1 min.

7.1.3.3. Incubate for 48 ± 2 h at 35 ± 1°C. If screening the enrichment broth with the BAX® system proceed with step 7.1.4 or 7.2 otherwise follow the isolation procedure of MFHPB-30 (see step 7.6).

7.1.4. Centrifugation and washing of the cells (Optional)

7.1.4.1 Transfer 1 mL of the BAX® system media to sterile DNase-free 1.5 mL centrifuge tubes and centrifuge for 3 min at 9,000 - 13,000 x g.

7.1.4.2. Discard the supernatant and resuspend the pellet in 1 mL of molecular grade water and vortex on high for 10 sec.

7.1.4.3. Centrifuge the tube again for 3 min at 9,000 - 13,000 x g. Discard the supernatant and resuspend the pellet in 0.1 mL molecular-grade water to concentrate the cells 10 fold.

7.1.4.4 Vortex the tube on high for 10 sec and add 5 μL of this suspension to a Bax lysis tube containing 0.2 mL of lysis reagent ( step 7.3.1.3.). Continue with the analysis from this step on.

7.2. Prepare equipment (Refer to BAX® System User Guide for details)

7.2.1. Make sure cooling blocks have been refrigerated overnight.

7.2.2. Warm up heating blocks. Check that temperatures are set to 55ºC and 95ºC.

7.2.3. Initialize BAX® system cycler/detector (perform verification, if prompted).

7.2.4. Create a rack file.

7.2.5. Select RUN FULL PROCESS in the menu bar to warm up the cycler/defector prior to placing your samples into the system.

7.3. Prepare Samples

7.3.1. Lyse samples

7.3.1.1. Arrange required number of lysis tubes in the rack according to the rack file. Prepare blank, positive and negative controls as per your labs requirements.

7.3.1.2. Prepare lysis reagent by pipetting 150 µL of protease into one 12 mL bottle of lysis buffer.

7.3.1.3. Add 200 µL of lysis reagent to each lysis tube.

7.3.1.4. Transfer 5 µL of enriched sample to the corresponding lysis tube.

7.3.1.5. Secure the caps and heat the tubes at 55°C for 60 minutes, then at 95°C for 10 minutes.

7.3.1.6. Place the lysis tubes in cooling block for at least 5 minutes.

7.3.2. Prepare samples for PCR

7.3.2.1. Place PCR tube holder onto PCR cooling block.

7.3.2.2. Arrange one PCR tube per sample in the holder.

7.3.2.3. Remove and discard lid from one strip of tubes at a time.

7.3.2.4. Using multi-channel pipette, transfer 50 µL of each lysed sample into a corresponding PCR tube.

7.3.2.5. Cover tubes with a new optical cap strip and secure tightly. Repeat for all samples.

7.3.2.6. Take the entire cooling block to the cycler/detector. Samples should remain in the cooling block until the cycler/detector is ready for loading, but no more than 30 minutes after tablet hydration.

7.4. Process samples

Follow the screen prompts of the PCR Wizard to load your samples, run the program and unload your samples, as specified in the User Guide.

7.5. Review Results

Screenshot - screen prompts of the PCR Wizard

Negative

Positive

Indeterminate

Signal Error

7.6. Isolation procedure or confirmation of presumptive positive results

Using the enrichment broth, presumptive positive results may be confirmed by immediately proceeding with the plating and confirmation steps of a cultural method (i.e. MFHPB-07 or MFHPB-30), as appropriate. Note that to isolate Listeria monocytogenes or other Listeria spp. from the enrichment broth, at least one chromogenic agar listed in MFHPB-30 must be used in addition to Oxford agar.

8. References

8.1 Pagotto, F., Hébert, K., J. Farber. 2011. Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples (MFHPB-30). In: Volume 2. The Compendium of Analytical Methods.

8.2 United States Department of Agriculture Food Safety and Inspection Service. Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry, Egg and Environmental Samples.

8.3 Warburton, D., Boville, A., Pagotto, F., Daley, E. and Chow, C. 2011. The Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples using palcam broth (MFHPB-07). In: Volume 2. The Compendium of Analytical Methods.

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