Detection of Salmonella by the Tecra Salmonella Visual Immunoassay™ (VIA) Method

Laboratory Procedure MFLP-35
June 2002

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Health Products and Food Branch
Ottawa

Evaluation Division
Bureau of Microbial Hazards
Food Directorate, Health Canada
Postal locator: 2204A1
Ottawa, Ontario, K1A 0L2

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

1. APPLICATION

This method is applicable to the detection of Salmonella from foods, food ingredients and environmental samples to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act.

The TECRA Salmonella Visual Immunoassay is an Enzyme-linked Immunosorbent assay (ELISA), which provides a rapid and specific screening system for the in vitro detection of Salmonella in food and environmental samples, after selective enrichment. Results are determined either visually or using a microtitre plate reader.

Presumptive positive results should be confirmed by standard cultural procedures. This is especially important in situations such as product recalls.

The TECRA Salmonella VIA has AOAC Official Methods approval for use with several different enrichment broths (AOAC methods 989.14 and 998.09). It also has approvals in several countries including France, Australia, New Zealand, Denmark and Brazil.

2. PRINCIPLE

The TECRA Salmonella Visual immunoassay is an Enzyme linked Immunoasorbent Assay (ELISA) performed in the "sandwich" configuration. High affinity "capture" antibodies specific for Salmonella have been adsorbed onto the surface of wells. If Salmonella antigens are present in the sample, they are captured by the antibodies. All other material in the sample is washed away. The "sandwich" is completed by the addition of enzyme labelled antibodies (conjugate) specific for Salmonella. The presence of Salmonella is indicated when the bound conjugate converts the substrate to a green color. Alternatively, if Salmonella is not present, no green color develops.

Total time to perform the test including selective enrichment and post enrichment in M broth is 42-50 hours.

If a presumptive positive Salmonella result is obtained with the VIA, this should be confirmed by streaking from the enrichment broth onto selective agar plates.

TECRA is the registered trademark of TECRA International Pty Ltd.13 Rodborough Rd, Frenchs Forest, NSW 2086 Australia.

3. DEFINITIONS OF TERMS

See Appendix A of Volume 3.

3.1 Sampling

See Appendix B of Volume 3.

3.2 Stringency of Sampling

See MFHPB-20 and implement if applicable.

3.3 "Routine" versus "Investigational" Sampling Plans

See MFHPB-20 and implement if applicable.

4. MATERIALS AND SPECIAL EQUIPMENT

4.1 Kit Components :

All reagents for the TECRA Salmonella Visual Immunoassay are provided in one kit, which contains enough material for a maximum of 94 tests (96 wells are provided but the positive and negative controls supplied with the kit must be run each time the test is performed). The kits are available in North America from International Bioproducts Inc. 21312 30^th Drive SE, Bothell WA 98021.

Instructions, sample record sheet
Plate holder
Resealing strip
Antibody coated microtitre wells
Reagent 1 - Wash concentrate
Reagent 2 - Positive control
Reagent 3 - Control diluent
Reagent 4 - Conjugate
Reagent 5 - Conjugate diluent
Reagent 6 - Substrate
Reagent 7 - Substrate diluent
Reagent 8 - Stop Solution

4.2 Additional Requirements :

35-37°C incubator
41-43°C incubator
Serological pipettes
Plastic squeeze bottle
Pipettes (20µL, 50 µL, and 200 µL)
Plastic wrap
Waterbath (100°C)
Small screw cap bottles or tubes capable of containing 5 mL and suitable for boiling.
Vortex mixer
Glass test tubes (15cm x 15cm)
Blender/stomacher/stomacher bags.

4.3 Media (VIA)

Media 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 8.2 for the formula of individual media.

Enrichment broth media:
Different enrichment protocols are described in the TECRA Methods Manual included in each kit.

Protocol 1: AOAC method 998.09 Option 1 RV(R10)/TT
Lactose broth or other non inhibitory broth as specified in FDA BAM (pre-enrichment)
Tetrathionate broth and Rappaport Vassiliadis (RV-R10) broth (selective enrichment)
M broth (post enrichment)

Protocol 2: AOAC Method 998.09 Option 2 RV(R10)
As for Option 1 but using Rappaport Vassiliadis (R10) broth only for selective enrichment

Protocol 3: AOAC Method 998.09 Option 3 TT
As for Option 1 but using Tetrathionate broth only for selective enrichment

Protocol 4: AOAC Method 989.14 TT/SC
Lactose broth or other non inhibitory broth as specified in FDA BAM (pre-enrichment)
Tetrathionate broth and Selenite Cystine broth (selective enrichment)
M broth (post enrichment)

Protocol 5: AFNOR approved method
Buffered Peptone Water (pre-enrichment)
Rappaport Vassiliadis (RV) broth and either Mannitol Selenite Cystine (MSC) or Selenite Cystine (SC)
broth (selective enrichment)
M broth (post enrichment)

5. PROCEDURE

5.1 Handling of Sample Units

5.1.1 Analyze samples as soon as possible. If necessary, store samples under time and temperature conditions that will prevent the growth or death of native microflora. If sample units have been abused in transit, resampling of the lot should be carried out.

1. Frozen Foods: Sample units that show no signs of thawing upon receipt may be stored in the freezer at - 10ºC to -20ºC.
2. Dried and shelf stable foods may be stored at room temperature.
3. Refrigerate all other foods, including those that are received in a partially thawed condition: analyze these samples as soon as possible preferably within 24 h of receipt.

5.1.2 Thaw frozen samples at room temperature within 60 min; if this is not possible, thaw the samples at refrigerator temperature (2 to 8ºC).

5.1.3 If the sample unit received for analysis is less than the recommended analytical unit, analyze the entire amount and record the weight used.

5.1.4 Blending of samples should be limited to the minimum time required to produce a homogeneous suspension. Excessive blending could result in physical damage that would adversely affect the viability of endogenous microflora.

For products that do not require blending, disperse the analytical unit into the appropriate preenrichment broth.

5.1.5 Use aseptic techniques and sterile equipment at all stages of analysis. Containment during the handling of powdered products is critical if cross-contamination of the work environment is to be avoided.

5.2 Enrichment

Samples should be enriched according to the appropriate enrichment protocol in the TECRA Salmonella Visual Immunoassay Methods Manual. Enrichment broths must be brought to room temperature (20-25°C) before combining with samples. When testing volumes greater than 225 mL, enrichment broths should be prewarmed before addition of samples.

5.3 Analysis

After post enrichment in M broth, samples should be heat killed and tested in the VIA kit in accordance with the TECRA Salmonella Visual Immunoassay Instruction Manual. The immunoassay will take approximately 2 hours and may be read visually or with the aid of a spectrophotometer.

5.4 Cultural Confirmation

Samples positive by the TECRA Salmonella Visual Immunoassay should be confirmed from the retained M broth or selective enrichment broth by streaking on standard selective agar and by standard biochemical and serological tests.

6. REFERENCES

6.1 Denise Hughes, Angela Daillianis and Louise Hill 1999. Salmonella in Foods - A New Enrichment Procedure for Use with the TECRA Salmonella Visual Immunoassay: Collaborative Study J.AOAC Int. 82: 634-647

6.2 International Commission on Microbiological Specifications for Foods (ICMSF).1986. Microorganisms in Foods 2. Sampling for microbiological analysis: Principles and Specific applications. Second edition. University of Toronto Press, Toronto, ON.

6.3 US Food and Drug Administration. 1995. Bacteriological Analytical manual (8th ed.) AOAC International, Gaithersburg, Md.