Detection of Listeria monocytogenes in foods by the VIDAS LMO 2™ method

Laboratory Procedure
MFLP-33
March 2012

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Health Products and Food Branch
Ottawa
Bureau of Microbial Hazards, Food Directorate
Postal Locator: 2204E
Health Canada, Ottawa ON K1A 0K9

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

1. Application

This method is applicable to the rapid detection of Listeria monocytogenes to determine compliance with the requirements of Section 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 except ready-to-eat meat and poultry, smoked fish, yogurt, kefir, and fermented dairy drinks. This method is not applicable to environmental samples as insufficient validation data have been received for this category. This revised method replaces MFLP-33, dated December 2011.

2. Principle

The VIDAS LMO 2 assay is an enzyme-linked fluorescent immunoassay (ELFA) for use on the VIDAS system for the qualitative detection of Listeria monocytogenes. The internal surface of the Solid Phase Receptacle (SPR), a pipette tip-like disposable device, is pre-coated during production with anti-L. monocytogenes antibodies. The VIDAS LMO 2 assay configuration prevents non-specific reactions with the SPR. Reagents for an assay are held in a sealed multi-chambered strip.

All assay steps are performed automatically and sequentially by the VIDAS instrument. The sample is inoculated into the reagent strip and cycled in and out of the SPR for a specific length of time. L. monocytogenes antigens present in the sample will bind to the anti-L. monocytogenes monoclonal antibodies, which are coated on the interior of the SPR. Unbound sample material is washed away. Antibodies conjugated with alkaline phosphatase are then cycled in and out of the SPR and react with the L. monocytogenes antigen-antibody complexes already adsorbed to the SPR wall. Unbound conjugate is removed by washing.

The fluorescent substrate, 4Bmethyl-umbelliferyl-phosphate, is then cycled in and out of the SPR where the enzyme conjugate converts the substrate to fluorescent 4-methyl-umbelliferone. The intensity of fluorescence is then measured at 450 nm.

VIDAS LMO 2™ is a registered Trademark of bioMérieux.

3. Definitions of terms

See Appendix A of Volume 3.

4. Collection of samples

See Appendix B of Volume 3.

5. Materials and special equipment

The following media (3-7) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 and reference 7.1 for the formula of individual media.

Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.

1. VIDAS LMO 2 Assay kit (Ref. 30704): contains the necessary reagents including standards and controls for the analysis of 60 test samples. The kit is available from bioMérieux Canada, Inc., 4535 Dobrin, St. Laurent, Québec H4R 2L8, Tel: (514) 336-7321, Fax: (514) 336-6450.
2. Large capacity VIDAS 30 or a reduced capacity miniVIDAS
3. Palcam broth
4. UVM 2
5. Palcam agar (or equivalent agar)
6. Oxford agar (or equivalent agar)
7. Chromogenic media (optional):
   • Rapid L. mono plates (Bio Rad Laboratories)
   • Aloa plates (AES Laboratoire)
8. Pipettor calibrated to dispense 500 μL and disposable tips
9. Stomacher and stomacher bags, blender or equivalent
10. Waterbath (95-100°C) or equivalent system
11. Incubators capable of maintaining 30°C and 35°C

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

6. Procedure

• 6.1 Handling of Sample Units
  • 6.1.1 In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.
  • 6.1.2 Analyze sample units as soon as possible after their receipt in the laboratory.
• 6.2 Preparation for Analysis
  • 6.2.1 Have sterile Palcam broth ready, pre-warmed to approximately 35°C.
  • 6.2.2 Clean the surface of the working area with disinfectant.
• 6.3 Preparation of Sample
  To ensure a representative analytical unit, agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit.
• 6.4 Primary Enrichment
  Add 25 g or mL of the food (the analytical unit) to 225 mL of Palcam broth in a blender jar or stomacher bag. For composite samples, maintain a ratio of 1 part sample material to 9 parts of Palcam broth (see Listeria policy for analytical unit sizes). Blend, stomach or vortex as required for thorough mixing and incubate at 35°C for 26 - 28 h.
• 6.5 Secondary Enrichment
  • 6.5.1 Agitate the Palcam Broth sufficiently to mix and allow large food particles to settle. Transfer 1 mL of the Palcam Broth to 9 mL of UVM 2. Incubate 26 (minimum) to 48 h at 30°C.
  • 6.5.2 Refrigeration of secondary enrichment cultures (UVM 2) - OPTIONAL
    
    The refrigeration of secondary enrichment cultures (UVM 2) provides for greater laboratory productivity and analytical flexibility. UVM 2 cultures arising on Friday from samples analyzed on the preceding Thursday are refrigerated over the weekend. On the following Monday, the contents of the refrigerated UVM 2 cultures are resuspended and proceed as described in 6.5.3 and 6.6.
  • 6.5.3 After incubation, vortex the UVM 2, allow particles to settle and proceed to step 6.6 It is NOT necessary to heat enrichment broths prior to testing in the VIDAS. Store the remaining enrichment broth at 2-8°C for later use in the confirmation of any VIDAS LMO 2 positive assay results.
• 6.6 Analysis
  • 6.6.1 Remove the VIDAS LMO 2 kit from the refrigerator. Remove necessary components from the kit and allow them to come to room temperature (minimum 30 min). Return all unused components to storage at 2-8°C.
  • 6.6.2 In the space provided, label LMO 2 Reagent Strips with the appropriate sample identification numbers.
  • 6.6.3 the applicable instructions pertaining to the entry of master lot, sample, standard and control data, according to the operational instructions which apply to the particular Vidas system you are using (the large capacity Vidas 30 or the miniVidas).
    
    
    Note: Components of different lots cannot be mixed and cannot be used past the expiry date.
  • 6.6.4 When a new kit lot is being used, specifications (i.e., the factory master calibration curve) must first be entered into the system using the master lot entry card included with each kit.
  • 6.6.5 The standard must be calibrated a minimum of every 14 days and when a new kit lot is being used. If a standard needs to be tested, enter AS1@ for the sample identification. The standard must be situated at the beginning of the Work List followed by the controls C1 and C2. The standard must be tested in duplicate, both with the same S1 designation, if it is to be stored in memory.
  • 6.6.6 Vortex standard, control and samples. Pipette 500 ± 50 µL of standard, control or sample into the center of the sample well of a LMO Reagent Strip.
  • 6.6.7 Load the LMO 2 Reagent Strips and LMO 2 SPRs into the positions that correspond to the VIDAS section indicated by the Work List. Check to make sure the color labels with the three letter assay code on the SPRs and the Reagent Strips match.
  • 6.6.8 Initiate the assay processing as directed in the VIDAS Operator's Manual. The assay will be completed in approximately 70 minutes.
  • 6.6.9 For further details, please refer to the package insert.
• 6.7 Interpretation
  Immunoassays producing relative fluorescence values (RFV) of ≥ 0.05 indicate the presumptive presence of Listeria monocytogenes in the test sample. All presumptive positive results must be confirmed culturally.
• 6.8 Confirmation
  • 6.8.1 Using the refrigerated secondary enrichment broth, proceed with plating and confirmation steps of a cultural method (i.e., MFHPB-07 or MFHPB-30).

7. References

• 7.1 Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.
• 7.2 Health Canada, Health Protection and Food Branch, Food Directorate. 2011. Policy on Listeria monocytogenes in Ready-to-Eat Foods.
• 7.3 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
• 7.4 A.M. Sewell, Warburton, D. W., Boville, A., Daley, E. F. and Mullen, K. 2003. A.Comparison of the Health Products and Food Branch and the Enzyme Linked Fluorescent Assay Methods for the Isolation and Identification of Listeria spp. from Foods. Int. J. Food Micro. (81)2:123-129.
• 7.5 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). In: Volume 2. The Compendium of Analytical Methods.

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