Detection of Listeria spp. in foods and environmental samples by the VIP method

Laboratory Procedure MFLP-34
February 2011

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Don Warburton and Ann Boville
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 raw meat, raw unprocessed seafood, raw frozen vegetables, cheese made from unpasteurized milk, and environmental samples. Insufficient validation data have been received for processed dairy, processed fish, ready-to-eat meats, and miscellaneous foods. This revised method replaces MFLP-34, dated January 2003.

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 fulfill MMC requirements (refer to Development of Methods in Volume 1 of the Compendium of Analytical Methods).

2. Description

VIP for Listeria is a visual immunoprecipitate assay that detects Listeria monocytogenes and related Listeria spp. It employs highly specific antibodies directed against Listeria antigens and has been specifically formulated to minimize cross-reactivity while maintaining superior sensitivity. The method has been modified by Warburton et al. (8.3; and unpublished data) to allow for the use of a single broth (Palcam Broth) as the pre-enrichment for all foods and environmental samples.

3. Principle

The VIP for Listeria method uses a proprietary reagent system to form an antigen-antibody- chromogen complex if Listeria is present. This ensures a high level of sensitivity and specificity to Listeria . Appropriately enriched samples are added to the VIP unit; any Listeria antigens will bind to the antibody-chromogen complex as it flows across a supporting membrane. When Listeria is present, the antigen-antibody-chromogen complex will form a detection line in the test sample window. Sample flow will continue down the membrane to form a control line in the test verification window regardless of whether the sample contains Listeria .

4. Definitions of terms

See Appendix A of Volume 3.

5. Collection of samples

See Appendix B of Volume 3.

6. Materials and special equipment

The following media (2-6) 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 8.1 for the formula of individual media.

1. VIP Listeria Assay Kit ( BioControl Systems Inc., website: www.biocontrolsys.com)
   
   
2. Palcam broth
   
   
3. UVM 2
   
   
4. Palcam agar (or equivalent agar)
   
   
5. Oxford agar (or equivalent agar)
   
   
6. Chromogenic media (optional):
   Rapid L. mono plates (Bio Rad Laboratories)
   Aloa plates (AES Laboratoire)
   
   
7. Pipettor calibrated to dispense 100 uL and disposable tips
   
   
8. Stomacher and stomacher bags, blender or equivalent
   
   
9. Waterbath (95-100 °C) or equivalent system
   
   
10. Incubators capable of maintaining 30 °C and 35 °C

7. Procedure

7.1 Handling of Sample Units

7.1.1 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.

7.1.2 Analyze sample units as soon as possible after their receipt in the laboratory.

7.2 Preparation of Sample / Primary Enrichment

7.2.1 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.

7.2.2 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). Quench the environmental swab in a 10 mL volume of a neutralizing agent and add this sample to 90 ml of Palcam Broth. Blend, stomach or vortex as required for thorough mixing and incubate at 35°C for 26 h. Environmental sponges may be added to 100 mL of Palcam Broth or composite up to 10 sponges with 100 mL of Palcam Broth for each sponge (see MFLP-41).

7.3 Secondary Enrichment

7.3.1 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.

7.3.2 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 7.3.3 and 7.4.

7.3.3 After incubation, transfer 2 mL of the enrichment broth into a tube and heat for 5 min in a waterbath at 100°C. Allow the tube to cool and then perform the VIP LIS test (7.4). Store the remaining enrichment broth at 2-8°C for later use in the confirmation of any VIP Listeria positive assay results.

7.4 VIP Assay Procedure

7.4.1 Open sealed pouch containing VIP units and remove required number of tests. One device is necessary for each test sample. VIP units may not be reused. Be certain to immediately reseal unused VIP units in pouch containing desiccant. Store at ambient temperature in a cool, dark location.

7.4.2 Gently shake the 2 mL aliquot of the heated secondary enrichment broth, then allow food particles to settle.

7.4.3 Transfer 0.1 mL of inoculated broth to sample additional well.

7.4.4 Incubate at ambient temperature for 10 minutes.

7.5 Reading Results

7.5.1 Examine VIP unit for the presence of a distinct line in the test verification window. This line should be dark in color when contrasted with the white background and extend across the window. Absence of a control line indicates an invalid test result. Contact BioControl Technical Services.

7.5.2 Observe test sample window. Presence of a distinct line, as described above, indicates a presumptive positive sample. Absence of a line is a negative. Differing intensities of test and control lines are acceptable as long as control line is present.

7.5.3 Positive and negative control cultures should be run to familiarize the analyst with results interpretation.

7.5.4 Autoclave units at 121°C for 15 minutes prior to discarding.

7.6 Confirmation of Positive VIP Samples

7.6.1 Using the refrigerated secondary enrichment broth, proceed with 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 in environmental samples, 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 for environmental samples.

8. References

8.1 Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.

8.2 Health Canada, Health Protection and Food Branch, Food Directorate. 2010. Policy on Listeria monocytogenes in Ready-to-Eat Foods.

8.3 A.M. 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/food-aliment

8.4 A.M. Sewell, Warburton, D. W., Boville, A., Daley, E. F. and Mullen, K. 2002. 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. In press.

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

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