MFLP-28: The Qualicon Bax® System method for the detection of Listeria monocytogenes in a variety of food

Laboratory Procedure MFLP-28
October 2011

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Microbiological Methods Committee
Microbiology Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, Health Canada
Postal Locator: 2204E
Ottawa, Ontario K1A 0K9

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

1. Application

This method is applicable to the detection of Listeria monocytogenes to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act.  Positive results must be confirmed with a cultural method. This method has been validated for use in all foods and environmental samples. This revised method replaces MFLP-28, dated February 2011. 

2. Description

The BAX® system is a convenient yes/no screening tool that uses 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 monocytogenes in a wide variety of foods. Following 24-48 hours primary enrichment and 18-24 hours secondary enrichment, sample preparation involves about 1 hour of user time, and the automated procedure delivers reliable results about 4 hours later. BAX® system validation studies used the USDA-FSIS enrichment method for meat, poultry and eggs; the AOAC method 993.12 for dairy; and the FDA-BAM method for other food types (see Section 8: Reference Methods). The BAX® system is designed for use by qualified lab personnel who follow standard microbiology practices.

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 monocytogenes, 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. Heating denatures or separates the DNA into single strands, then as the mixture cools, primers recognize and anneal to the target DNA sequence. DNA polymerase then uses nucleotides to extend the primers, thereby creating two copies of the target DNA fragment. Repeated cycles of denaturing, annealing, and extending produce exponential increases in the number of target DNA fragments within a matter of hours. If the target sequence is not present, no detectable amplification takes place.

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, eliminating 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

Note: The Laboratory Supervisor must ensure that the analysis described in this method is carried out in accordance with the International Standard referred to as "ISO/IEC 17025:2005 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories".

Note: It is the responsibility of each laboratory to ensure that the temperature of the incubators or water baths is maintained at the recommended temperatures.  Where 35°C is recommended in the text of the method, the incubator may be at 35 +/-1.0ºC.  Similarly, lower temperatures of 30 or 25°C may be +/- 1.0ºC.  However, where higher temperatures are recommended, such as 43 or 45.5ºC, it is imperative that the incubators or water baths be maintained within 0.5ºC due to potential lethality of higher temperatures on the microorganism being isolated.

6.1 Supplied with kit - (no.17710609; sufficient for 96 tests - DuPont Qualicon, phone: 1-800-863-6842, fax: 302-695-5301)

2 bags PCR sample 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.

1 bag Optical caps, 12 strips of 8 caps.

2 bottles Lysis buffer, pH 8.35 ± 0.05 @ 25 °C. 12 ml/bottle. Used to prepare working lysis reagent.

1 vial Protease solution, 400 μL/vial. Used to prepare working lysis reagent

6.2 Enrichment broths
Primary enrichment - varies by food type (see Section 7)
UVM broth
Demi-Fraser broth

6.3 Secondary enrichment
MOPS-BLEB

6.4 Equipment
Stomacher
Incubator
Microcentrifuge
Vortex

6.5 Other (included with BAX® system start-up package)
Cycler/detector with verification plates
Computer workstation with Microsoft Windows ® operating system, BAX® system application, and printer
Dry block heaters with thermometers inserts for lysis tubes
Capping/decapping tools
Cooling block with inserts for lysis tubes and PCR tubes
BAX® system User Guide

6.6 Additional Supplies
PCR tubes
Lysis tubes with caps and rack
Tips for pipettes
Powder-free nitrile gloves
PCR tube holders
Molecular grade water (sterile and DNase free)
Various pipettes for reagent and sample transfers

7. Procedure

7.1 Collect and enrich samples

Note:  To allow flexibility for incubation times stated, the following guidelines can be used. Incubation times of 24 h are " 2h; incubation times of 48 h are " 4 h.

Food Samples: Add 25 g or mL of the food (analytical unit) to 225 ml of enrichment broth (refer to the table below) in a sterile container. For composite samples, analytical units may be combined up to 125g or mL (e.g., 125 g or mL in 1125 mL of enrichment broth). If alternate analytical units are required, maintain a ratio of 1 part sample material to 9 parts enrichment broth.

For both environmental samples and food, blend, stomach or vortex as required for thorough mixing. The enrichment broth may be incubated in the stomacher bag or other sterile container.

Sample Type	Primary enrichment Pre-warm at 30 ºC ±1 ºC	Secondary Enrichment
Raw Meat and Poultry Products	Weigh 25 g sample into sterile container.  Homogenize with 225 mL Demi Fraser broth.  Incubate 24 h at 30°C.	After 24 h of incubation mix the primary enrichment well, and then transfer 0.1 mL of the enrichment to 9.9 mL MOPS-BLEB.  Incubate 18-24 h at 35°C.
Ready-to-eat Meat and Poultry	Weigh 25 g sample into sterile container.  Homogenize with 225 mL Enrichment LEB (UVM1) broth.  Incubate 48 h at 30 ºC.	After 48 h of incubation mix the primary enrichment well, and then transfer 0.1 mL of the enrichment to 9.9 mL MOPS-BLEB.  Incubate 18-24 h at 35°C.
Fruits and Vegetables
Fish and Seafood Products (including smoked fish)
Dairy Products
Other Foods
Environmental Swabs	Add the environmental sponge or large swabs to 100 mL of LEB (UVM1) or composite up to 10 sponges with 100 mL of LEB per sponge (see MFLP-41). Place smaller environmental swabs (e.g., cotton tip) in 10 mL of LEB or composite up to 10 swabs with 10 mL LEB per swab. Incubate 48 h at 30 ºC.	After 48 h of incubation mix the primary enrichment well, and then transfer 0.1 mL of the enrichment to 9.9 mL MOPS-BLEB. Incubate 18-24 h at 35°C.

Proceed to step 7.2 or continue with step 7.1b below.

7.1b Centrifugation and washing of the cells (The optional centrifugation step applies only to RTE meat)

• 7.1.1 Transfer 1 ml of MOPS-BLEB to sterile DNase-free 1.5ml microfuge tubes and centrifuge for 3 min at 9,000-13,000 x g.
• 7.1.2 Discard the supernatant and resuspend the pellet in 1 ml of molecular-grade water and vortex on high for 10 sec.
• 7.1.3 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 Vortex the tube on high to completely resuspend the pellet and add 5ul of this suspension to a BAX Lysis tube containing 0.2mL of lysis reagent (step 7.3.1.4). Continue with the analysis form this step onwards.

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/detector.

7.3 Prepare samples

• 7.3.1 Lyse samples
  • 7.3.1.1 Arrange required number of lysis tubes (one for each sample and one for the "blank") in the rack according to the rack file.
  • 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

7.6 Confirm presumptive positive results

Using the secondary enrichment broth, proceed with the plating and confirmation steps of a cultural method (i.e. MFHPB-07 or MFHPB-30).

8. References

8.1 Official Methods of Analysis (2002), 17th edition. AOAC INTERNATIONAL, Gaithersburg, MD, section 993.12.

8.2 Pagotto, F., Hébert, K., and J. Farber. 2011. Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples (MFHPB-30). Volume 2. The Compendium of Analytical Methods. http://www.hc-sc.gc.ca/food-aliment

8.3 US Department of Agriculture, Food Safety and Inspection Service, Office of Public Health and Science. Microbiology Laboratory Guidebook [Internet]. Washington: The Dept; c2002 [rev 2002 Apr 29; cited 2002 July 22]. Chapter 8, Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry, Egg and Environmental Samples. Available from  http://www.fsis.usda.gov/OPHS/microlab/mlgchp803.pdf

8.4 US Food & Drug Administration, Center for Food Safety & Applied Nutrition. Bacteriological Analytical Manual Online [Internet]. Washington: The Admin; c2001 [rev 2001 April; cited 2002 July 22]. Chapter 10, Listeria monocytogenes. Available from  http://www.cfsan.fda.gov/~ebam/bam-10.html

8.5 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). Volume 2. The Compendium of Analytical Methods. http://www.hc-sc.gc.ca/food-aliment

8.6 Microbiological Methods Committee. 2010. Environmental sampling for the detection of microorganisms (MFLP-41). Volume 3: The Compendium of Analytical Methods. http://www.hc-sc.gc.ca/food-aliment

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