Detection of Listeria spp. in foods and environmental samples by the VIDAS Listeria^TM method

HPB Method
MFHPB-29
March 2012

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Health Products and Food Branch
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 spp.to determine compliance with the requirements of Section 4 and 7 of the Food and Drugs Act. This method has been validated for use in environmental samples and all foods except ready-to-eat meat and poultry, smoked fish, and yogurt. This revised method replaces MFHPB-29, dated December 2011.         

Note:While this method is only approved for certain food products, as listed above, it is assumed that this method could be used with other foods. To ensure the method is fit for purpose for commodities outside the application, it is imperative that other commodities be properly validated following the criteria in the Compendium of Analytical Methods. It is requested that these validation data be forwarded to the Microbiological Methods Committee so the Application Section can be expanded to include these new foods if the data fulfil MMC requirements (refer to Development of Methods in Volume 1 of the Compendium of Analytical Methods).

2. Principle

The VIDAS LIS assay is an enzyme-linked fluorescent immunoassay (ELFA) for use on the VIDAS system for the qualitative detection of Listeria spp. The internal surface of the Solid Phase Receptacle (SPR), a pipette tip-like disposable device, is pre-coated during production with anti-Listeria antibodies. The VIDAS LIS 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. Listeria antigens present in the sample will bind to the anti-Listeria 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 Listeria antigen-antibody complexes already adsorbed to the SPR wall. Unbound conjugate is removed by washing.

The fluorescent substrate, 4-methyl-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 LISTM is a registered Trademark of bioMérieux.

3. Definitions of terms

See Appendix A of Volume 2.

4. Collection of samples

See Appendix B of Volume 2.

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 2 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 Listeria Assay kit (Ref. 30 700): 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)
   Agar for Listeria according to Ottaviani & Agosti (ALOA)
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 is 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

Food samples: 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).

Environmental Samples: Add the environmental sponge or large swabs to 100 mL of Palcam broth or composite up to 10 sponges with 100 mL Palcam broth per sponge (see MFLP-41). Place smaller environmental swabs (e.g., cotton tip) in 10 mL portions of Palcam broth or composite up to 10 swabs with 10 mL Palcam broth per swab.

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 Wednesday or 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, transfer 2 mL of the enrichment broth into a tube and heat for 15 +/- 2 min in a waterbath at 95-100°C. Allow the tube to cool and then perform the VIDAS LIS test. Store the remaining enrichment broth at 2-8°C for later use in the confirmation of any VIDAS Listeria positive assay results.

6.6 Analysis

6.6.1 Remove the VIDAS LIS 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 LIS Reagent Strips with the appropriate sample identification numbers.

6.6.3 Follow 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 "S1" 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 boiled samples. Pipette 500 +/- 50 μL of standard, control or boiled sample into the center of the sample well of a LIS Reagent Strip. It is essential that all enrichment broths be heated for 15 minutes at 95-100°C and cooled to room temperature prior to testing on VIDAS.

6.6.7 Load the LIS Reagent Strips and LIS SPRs into the positions that correspond to the VIDASsection 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 45 minutes.

6.6.9 For further details, please refer to the package insert.

6.7 Interpretation

Immunoassays producing relative fluorescence values (RFV) of ≥ 0.10 indicate the presumptive presence of Listeria spp. in the test sample.

6.8 Confirmation

6.8.1 Using the refrigerated secondary enrichment broth, presumptive positive results may be confirmed with the plating and confirmation steps of a cultural method (i.e. MFHPB-07 or MFHPB-30), as appropriate.

Note: If the results indicate that Listeria spp. have not been detected, it is understood that the presence of L. monocytogenes has also not been detected, since no species belonging to the Listeria genus were detected. However, if Listeria spp. are detected, regardless of the amount of colony screening or additional confirmation steps done, these results cannot be used to state that L. monocytogenes is absent. Report all species of Listeria that are identified.

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. 2010. Policy on Listeria monocytogenes in Ready-to-Eat Foods. http://www.hc-sc.gc.ca/fn-an/legislation/pol/policy_listeria_monocytogenes_2011-eng.php
• 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. http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php
• 7.4 Sewell, A.M., 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. http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php

Acknowledgements

The following people are thanked for their assistance in the development of this method:

• Cindy Chow and Sheila Davidson of HC, Ottawa
• Lorraine Gour, Ghislaine Bélanger and Gilles Brunelle of HC, Longueuil
• Yvon-Louis Trottier and Lyne Laflamme of CFIA, St-Hyacinthe
• Karen Jessett, George Huszczynski and Nelly Denis of CFIA, Mississauga
• Kim Hopkins, Silliker Labs, Toronto, Ontario
• and Karen Mullen, BioMerieux Canada, Scarborough, Ontario.

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