Detection of Listeria monocytogenes and other Listeria spp. in food products and environmental samples by the VIDAS^® Listeria species Xpress (LSX) method

Laboratory Procedure
MFLP-77
October 2012

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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 rapid detection of Listeria monocytogenes and other 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 raw meats, dairy products, fruits and vegetables, fish and seafood, environmental samples and the food type heat processed ready-to-eat (RTE) meat and poultry. This revised method replaces MFLP-77, dated June 2012.

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 validation data be forwarded to the Microbiological Methods Committee so the Application Section can be expanded to include these new foods if the data comply with MMC requirements (refer to Development of Methods in Volume 1 of the Compendium of Analytical Methods).

2. Principle

VIDAS^® LSX is an enzyme-linked fluorescent immunoassay (ELFA) for use on the VIDAS^® instrument for the detection of Listeria antigens using the ELFA technique. The internal surface of the Solid Phase Receptacle (SPR^®), a pipette tip-like disposable device, is pre-coated during kit production with anti-Listeria antibodies. Reagents for an assay are ready-to-use and 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^® several times. Listeria antigens present in the sample will bind to the anti-Listeria antibodies coating 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 will bind with the Listeria antigen-antibody complexes already adsorbed to the SPR^® wall. Unbound conjugate is removed by further wash steps.

The fluorescent substrate, 4-methyl-umbelliferyl-phosphate, is cycled in and out of the SPR^®. The conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4-methyl-umbelliferone), the fluorescence of which is measured at 450 nm. A test value is generated for each sample and this value is compared to internal references (thresholds) and each result is interpreted (positive, negative).

3. Definition of Terms

See Appendix A of Volume 3.

4. Collection of Samples

See Appendix B of Volume 3.

5. 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"(7.3).

Note: It is the responsibility of the laboratory to ensure equivalency if any variations of the media formulations listed here are used (either product that is commercially available or made from scratch). Please forward equivalency data to the Editor of Compendium of Analytical Methods for consideration of modification of this method.

Mandatory media and equipment (from bioMerieux Canada, Inc. 1-800-361-7321)

• LX broths (bags, bottles or tubes)
• VIDAS^® family instrument (mini-VIDAS or VIDAS 30)
• VIDAS^® LSX kit (ref. 30224): contains the necessary reagents including standards and controls for the analysis of 60 test samples.
• VIDAS^® Heat and Go, or water bath (95-100°C) or equivalent system

Other materials and equipment

• Stomacher and stomacher bags with filter, blender or equivalent
• Pipette calibrated to dispense 500 µl and disposable tips
• Incubators capable of maintaining 30°C

Note: It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures. Where 35°C is recommended in text of the method the incubator may be at 35 ± 1.0° C. Similarly, lower temperatures of 30 or 25 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 water baths be maintained within 0.5°C due to potential lethality of the higher temperatures on the microorganism(s) being isolated.

6. Procedure

6.1 Handling of Sample Units

6.1.1 During storage and transport, with the exception of shelf-stable products, keep the sample units refrigerated.  Keep sample units of frozen products frozen. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.

6.1.2 Analyze the sample units as soon as possible after receipt at the laboratory.

6.2 Preparation for Analysis

6.2.1 Have sterile LX broth available, pre-warmed to 30 ± 2°C before use.

6.2.2 Clean the surface of the working area with a suitable 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

6.4.1 For food samples, aseptically add 25 g or mL of sample (analytical unit) to 225 mL of LX broth in a stomacher bag. Blend, stomach or vortex as required for thorough mixing.

6.4.2 For environmental samples, aseptically add 10 mL of LX broth for each swab or 100 mL of LX broth for each sponge, or prepare a 1/10 ratio sample with LX broth for other sample types.  Stomach, vortex, or homogenize as required for thorough mixing.

6.4.3 For composited samples, maintain a ratio of 1 part sample material to 9 parts of LX broth.

6.4.4 Incubate at 30 ± 1°C for 22 - 24 hours for food samples or 24 - 26 hours for environmental samples.

6.5 Secondary Enrichment

Only for food samples, after incubation, mix and transfer 3 mL of the suspension into 6 mL of LX broth. Incubate for 6 - 24 hours at 30 ± 1°C.

6.6 Analysis

6.6.1 Remove the VIDAS^® LSX kit from the refrigerator. Only remove the required reagents needed and allow them to come to room temperature. Use one LSX strip and one LSX SPR^® for each sample, control or standard to be tested. Make sure the storage pouch has been carefully resealed after the required SPRs have been removed.

Note: Components of different lots cannot be mixed and cannot be used past the expiry date.

Note: When a new kit lot is being used, specifications (i.e., the factory master calibration curve data) must first be entered into the system using the master lot entry card included with each kit. Calibration, using the standard provided in the kit, must be performed for each lot of reagents upon opening, and every 14 days thereafter. Please refer to the package insert for the VIDAS^® PTC protocol data entry, master lot data entry and calibration instructions.

6.6.2 After incubation, homogenize the LX broth.

6.6.3 Prepare the sample for the VIDAS assay by either using a water bath or the VIDAS^® Heat and Go Device.

6.6.4 If a water bath is used, transfer 1-2 mL of LX broth into a tube and seal. Heat for 15 ± 1 minutes at 95-100°C. Cool the tube. Mix the boiled broth and transfer 0.5 mL into the sample well on the VIDAS^® strip. In the space provided, label the strip with the appropriate sample identification number.

6.6.5 If the VIDAS^® Heat and Go device is used, transfer 0.5 mL of the LX broth into the sample well on the strip. Label the strip and heat for 15 ± 1 minutes (refer to the user's manual). Remove the strip and allow to come to room temperature.

Note: Do not heat the standard or the controls.

6.6.6 Initiate the assay as directed in the VIDAS^® operator's manual. Insert the SPR^® and strips into the positions that corresponds to the VIDAS^® section indicated by the work list. Check to make sure the color labels with the assay code on the SPR^® and the reagent strips match.

6.7 Interpretation

Once the assay is completed, the computer analyzes results automatically. Immunoassays producing relative fluorescence values (RFV) of ≥ 0.1 indicate the presumptive presence of Listeria spp. in the test sample.

6.8 Confirmation

Using the unheated secondary enrichment broth, presumptive positive results for Listeria spp. may be confirmed by proceeding with the plating and confirmation steps of a cultural method (i.e. MFHPB-07 or MFHPB-30), as appropriate. For confirmation of the presence of L. monocytogenes, or to confirm the absence of L. monocytogenes in the presence of other Listeria species, a chromogenic medium (as per MFHPB-30) that differentiates L. monocytogenes from other Listeria spp. must be used with the plating and confirmation steps of a cultural method (i.e., MFHPB-07 or MFHPB-30).

7. References

7.1 EN ISO 11290-1/A1 - 2005. Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes - Part 1: Detection method.

7.2 EN ISO 16140 - 2003. Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods.

7.3 ISO/IEC 17025:2005. General requirements for the competence of testing and calibration laboratories.

7.4 Johnson R., Jechorek R. 2011. Evaluation of VIDAS^® Listeria species Xpress (LSX) Immunoassay Method for the Detection of Listeria species in Foods: Collaborative Study. J of AOAC INTERNATIONAL Vol. 04, no. 1, pp. 159-171.

7.5 Pagotto, F., K. Hébert, and, J. Farber. 2011. Isolation of Listeria monocytogenes from all Foods and Environmental Samples (MFHPB-30). The Compendium of Analytical Methods. In: Volume 2. The Compendium of Analytical Methods.

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