Detection of Staphylococcal Enterotoxins in Food Products Using the Vidas® Staph Enterotoxin II (set2), an Elfa (Enzyme Linked Fluorescent Assay) Technique

MFLP-65
April 2008

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Health Products and Food Branch
Ottawa

Don Warburton
and the Microbiological Methods Committee
Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Postal Locator: 2204E
HPFB, Ottawa, Ontario, K1A 0L2

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

1. Application

VIDAS® Staph enterotoxin II is an automated qualitative test for use on the VIDAS analyzer for the detection of staphylococcal enterotoxins in food products (canned food, liquid products, dehydrated products, dairy products, meat, delicatessen and seafood products (AOAC n°0070404)) using the ELFA technique (Enzyme Linked Fluorescent Assay). The VIDAS SET2 assay provides a direct method for screening food for the presence of any of the seven staphylococcal toxins.

2. Principle

VIDAS Staph enterotoxin II is an enzyme-linked fluorescent immunoassay (ELFA) used on the automated VIDAS instrument (see the Operator's Manual) for the specific detection of staphylococcal enterotoxins. The Solid Phase Receptacle (SPR®) serves as the solid phase as well as the pipetting device for the assay. The interior of the SPR is coated with monoclonal anti-staphylococcal enterotoxin antibodies. Reagents for the assay are ready-to-use and pre-dispensed in the sealed reagent strips. All of the assay steps are performed automatically by the instrument. An aliquot of food extract is placed into the reagent strip by the user and is then cycled in and out of the SPR for a specific length of time. Staphylococcal enterotoxins present in the sample will bind to the anti-SET monoclonal antibodies which are coated on the interior of the SPR. Unbound sample components are washed away. Alkaline phosphatase-labeled antibodies are cycled in and out of the SPR and will bind to any SET captured on the SPR wall. Further wash steps remove unbound conjugate. During the final detection step, the substrate (4-Methylumbelliferyl phosphate) is cycled in and out of the SPR. The bound enzyme conjugate catalyzes the hydrolysis of this substrate into a fluorescent product (4-Methylumbelliferone), the fluorescence of which is measured at 450 nm. When the assay is completed, the results are analyzed automatically by the instrument which generates a test value and prints a report for each sample. This value is compared to a threshold and each result is interpreted (positive, negative).

3. Definition of Terms

See Appendix A of Volume 2.

4. Collection of Samples

See Appendix B of Volume 2.

5. Materials and Special Equipment

1. VIDAS SET2 (Ref 30 705) Assay kit contains the necessary reagents including standards and controls for the analysis of 30 test samples. The kit is available from bioMérieux Canada, Inc., 7815 Henri-Bourassa West, St. Laurent, Québec H4S 1P7. Tel: (514) 336 7321; Fax: (514) 336 6450.
   
   
2. A large capacity VIDAS 30 or a reduced capacity mini VIDAS
   
   
3. Pipette with disposable tip calibrated to dispense 500 µl
   
   
4. Blender or Stomacher® for 400 mL Stomacher bag
   
   
5. Centrifugation tubes (50 mL)
   
   
6. Syringes (20 mL)
   
   
7. pH paper
   
   
8. Trichloroacetic acid (TCA) 90% (5.5N)
   
   
9. TRIS buffer (0.3M pH 8.0)
   
   
10. Sodium hydroxide 1N and 4 N
    
    
11. Hydrochloric acid 5N

6. Procedure

Carry out the test in accordance with the following instructions:

6.1 Reagents preparation and Sample Pre-treatment

6.1.1 Preparation of the extraction buffer:

Dilute the whole concentrated extraction buffer provided in the kit with sterile distilled water to obtain 1 liter of ready-to-use solution. Homogenize. Store at 2-8°C for up to 3 months. The concentrated buffer can also be diluted in different volumes depending on frequency of use (dilution rate = 1/18).

6.1.2 Preparation of the 90% trichloroacetic acid solution:

Dissolve 90 g of trichloroacetic acid in 40 mL of demineralized water. Adjust the final volume to 100 mL using demineralized water. The solution can be stored for 1 month at 18-25°C.

6.1.3 pH adjustment of food extracts:

For pH adjustment of food extracts we recommend the use of a strip paper with 3 color bands and a precision at least equal to 0.5 pH unit.

6.2 Sample Preparation

6.2.1 General extraction protocol:

• 6.2.1.1 To 25 g of food add 25 mL of reconstituted extraction buffer diluted as indicated previously, in a Stomacher bag.
  
  
• 6.2.1.2 Blend at high speed for 3 minutes to obtain a homogeneous suspension.
  
  
• 6.2.1.3 Let stand for 15 minutes at 18-25°C.
  
  
• 6.2.1.4 Centrifuge the blended sample in the extraction solution for 15 minutes at 3000-5000 g at 18-25°C.
  
  
• 6.2.1.5 Pump the supernatant through a moistened absorbent cotton placed in a syringe, using the plunger
  
  
• 6.2.1.6 Check the filtrate pH and adjust it to between 7.5 and 8.0 if necessary, using 1N NaOH.
  
  
• 6.2.1.7 Recover 500 µl of the filtrate and place in the sample well of a VIDAS SET2 Reagent strip before initiating the assay.

6.2.2 Liquid food:

• 6.2.2.1 Dilute the concentrated food product as indicated by the manufacturer.
  
  
• 6.2.2.2 Readjust the pH if necessary to between 7.5 and 8.0, using 1N NaOH.
  
  
• 6.2.2.3 In the case of a precipitate, centrifuge and filtrate the suspension as described in the general extraction protocol.
  
  
• 6.2.2.4 Recover 500 µl of the filtrate and place in the sample well of a VIDAS SET2 Reagent strip before initiating the assay.

6.2.3 Dehydrated food:

• 6.2.3.1 Rehydrate the food product with an equivalent volume of distilled water or according to the manufacturer's instructions.
  
  
• 6.2.3.2 Leave the rehydrated sample for one hour at room temperature.
  
  
• 6.2.3.3 Weigh 25 g of rehydrated food and add 25 mL of reconstituted extraction buffer.
  
  
• 6.2.3.4 Proceed then as described in the general extraction protocol (for NFDM), proceed as described in protocol 6.2.6 Dairy products).

6.2.4 Canned food (post retort contamination):

• 6.2.4.1 Blend the whole canned food or a representative aliquot to obtain a homogeneous suspension.
  
  
• 6.2.4.2 To 25 g of food add 25 mL of reconstituted extraction buffer.
  
  
• 6.2.4.3 Proceed then as described in the general extraction protocol.

6.2.5 Raw meat products, seafood and delicatessen meats:

• 6.2.5.1 Blend 25 g of food in 25 mL of distilled water at high speed for 3 minutes to obtain a homogeneous suspension. If the suspension is too dense add an additional 25 mL of distilled water and re-blend.
  
  
• 6.2.5.2 Recover the whole extract.
  
  
• 6.2.5.3 Check the pH and adjust it to 4.0 using 5N HCL.
  
  
• 6.2.5.4 Let stand for 15 to 30 minutes at 18-25°C.
  
  
• 6.2.5.5 Centrifuge the blended sample in the extraction solution for 15 minutes at 3000-5000 g at 18-25°C.
  
  
• 6.2.5.6 Pump the supernatant through a moistened absorbent cotton placed in a syringe, using the plunger.
  
  
• 6.2.5.7 Check the filtrate pH and adjust it to between 7.5 and 8.0 if necessary, with 1N NaOH.
  
  
• 6.2.5.8 In the case of a precipitate, centrifuge an aliquot as described previously.
  
  
• 6.2.5.9 Recover 500 µl of the filtrate and place in the sample well of a VIDAS SET2 Reagent strip before initiating the assay.

6.2.6 Dairy products:

• 6.2.6.1 Protocol without concentration:
  
  
  • 6.2.6.1.1 To 25 g of food add 40 mL of distilled water pre-incubated at 38 ± 2°C.
    
    
  • 6.2.6.1.2 Blend at high speed for 3 minutes to obtain a homogeneous suspension.
    
    
  • 6.2.6.1.3 Let stand for 30 minutes at 18-25°C.
    
    
  • 6.2.6.1.4 Check the pH and adjust it to between 3.5 and 4.0 using 5N HCL.
    
    
  • 6.2.6.1.5 Centrifuge this suspension for 15 minutes at 3000-5000 g at 18-25°C.
    
    
  • 6.2.6.1.6 Recover the supernatant and adjust the pH to between 7.5 and 8.0 using 1N NaOH.
    
    
  • 6.2.6.1.7 Centrifuge for 15 minutes at 18-25°C at 3000-5000 g and filter if necessary.
    
    
  • 6.2.6.1.8 Recover 500 µl of the filtrate and place in the sample well of a VIDAS SET2 Reagent strip before initiating the assay.

• 6.2.6.2 For liquid products (i.e. milk):
  
  
  • 6.2.6.2.1 Adjust the pH of 25 mL (or 25 g) of the product to between 3.5 and 4.0 using 5N HCL.
    
    
  • 6.2.6.2.2 Proceed then as previously described.
    
    
• 6.2.6.3 Protocol using Trichloroacetic acid concentration (TCA):

Note:
This protocol is recommended for increasing the concentrations of the toxins for all dairy products and suppressing rare interference found with some raw milk cheeses (e.g Roquefort).

Caution:
Do not use metal spoons or utensils with trichloroacetic acid, please refer to the MSDS sheet.

• • 6.2.6.3.1 The method of preparation for the trichloroacetic acid solution is described in the section "Preparation of the 90% trichloroacetic acid solution" (see Section 6.1.2).
    
    
  • 6.2.6.3.2 Proceed as described in the dairy product protocol until the end of the first centrifugation.
    
    
  • 6.2.6.3.3 Recover the supernatant in a centrifugation tube and measure its volume V.
    
    
  • 6.2.6.3.4 Rapidly add to the supernatant V a volume Y of a TCA solution (at 90% in water) to obtain a 5% final concentration: Y = V x 5/100.
    
    
  • 6.2.6.3.5 Homogenize and precipitate the proteins for 30 minutes at 18-25°C.
    
    
  • 6.2.6.3.6 Centrifuge for 30 minutes at 3000-5000g at 18-25°C.
    
    
  • 6.2.6.3.7 Carefully discard the supernatant.
    
    
  • 6.2.6.3.8 Dissolve the protein pellet in a volume of TRIS 0.3M pH 8.0 corresponding to 1/10 of the starting volume V of the supernatant.
    
    
  • 6.2.6.3.9 Adjust the pH to between 7.5 and 8.0 using 4N NaOH.
    
    
  • 6.2.6.3.10 At this pH, the milky solution becomes clear. If the solution shows particles in suspension, centrifuge again for 15 minutes at 3000-5000g at 18-25°C.
    
    
  • 6.2.6.3.11 Recover 500 µl of the filtrate and place in the sample well of a VIDAS SET2 Reagent Strip before initiating the assay.

6.3 Extract storage

The VIDAS SET2 assay must be performed immediately after extraction. Extracts of food products other than dairy products, may be stored for 7 days at -25 ± 6°C. It is recommended that specific matrices be validated for stability of extracts when stored at -25 ± 6°C for 7 days.

6.4 VIDAS Analysis

6.4.1 Master lot data entry and Calibration

Refer to the VIDAS SET 2 package insert for more information.

6.4.2 Procedure for Analysis

• 6.4.2.1 Only remove the required reagents from the refrigerator and allow them to come to room temperature for at least 30 minutes.
  
  
• 6.4.2.2 Use one VIDAS "SET2" strip and one VIDAS "SET2" SPR for each sample, control or calibrator to be tested. Make sure the storage pouch has been resealed after the required SPRs have been removed.
  
  
• 6.4.2.3 Type or select "SET2" on the instrument to enter the test code. The standard must be identified by "S1", and tested in duplicate. If the positive control is to be tested, it should be identified by "C1". If the negative control needs to be tested, it should be identified by C2. Note: If a standard is being tested, type "S" ("S" then "1" on mini VIDAS) for the sample identification.
  
  
• 6.4.2.4 Thoroughly mix the standard, controls and samples before use in order to improve result reproducibility. Accurately pipette 500 µl of standard when performing a calibration to ensure correct measurement.
  
  
• 6.4.2.5 Pipette 500 µl ± 50 µl of sample, or control into the sample well (Note: samples and controls must be tested singly).
  
  
• 6.4.2.6 Insert the SPRs and strips into the instrument. Check to make sure the color labels with the assay code on the SPRs and the Reagent Strips match.
  
  
• 6.4.2.7 Initiate the assay as directed in the Operator's Manual. All the assay steps are performed automatically by the instrument. The assay will be completed within approximately 80 minutes.
  
  
• -6.4.2.8 After the assay is completed, remove the SPRs and strips from the instrument.
  
  
• 6.4.2.9 Dispose of the used SPRs and strips into an appropriate receptacle.

6.5 Reading Results

6.5.1 Once the assay is completed, results are analyzed automatically by the computer.

6.5.2 The RFV (Relative Fluorescence Value) is calculated by subtracting the background reading from the final result. This calculation appears on the result sheet.

6.6 Interpretation of results

6.6.1 The RFV obtained for each sample is interpreted by the VIDAS system as follows:

Test value="Sample" RFV / Standard RFV

Test value="Sample" RFV / Standard RFV

Test Value	Interpretation
< 0.13	Negative
= 0.13	Positive

6.6.2 A result with a test value that is less than the threshold value indicates that the sample does not contain staphylococcal enterotoxin or contains staphylococcal enterotoxin at a concentration below the detection limit.

6.6.3 A result with a test value that is greater than or equal to the threshold value indicates a sample contaminated with staphylococcal enterotoxin.

7. Notes

7.1 Quality Control

One positive control and one negative control are included in each VIDAS SET2 kit.

These controls must be performed immediately after opening a new kit to ensure that reagent performance has not been altered.

Controls must be tested during each calibration.

The instrument will only be able to check the control values if they are identified by C1 and C2.

The expected value for the positive control is indicated on the MLE card (index).

Results cannot be validated if the positive control value deviates from the expected values.

It is the responsibility of the user to perform Quality Control in accordance with any local applicable regulations.

7.2 Limitations of the method

Refer to the package insert for more information on levels of detection.

Any change or modification in the procedure may affect the results. Rare interference has been observed with some blue veined-type raw milk cheeses (e.g. Roquefort). The trichloroacetic acid concentration method enables the suppression of this interference.

8 REFERENCES

8.1 Adesiyum, A.A., S.R. Tatini, and D.G. Hoover. 1984. Production of enterotoxin(s) by Staphylococcus hyicus. Vet. Microbiol. 9:487-495.

8.2 Bergdoll, M.S. 1983. Enterotoxins I : Staphylococci and Staphylococcal infections. Volume 2. Eds: Easmon C. and C. Adlam. Academic Press. New York, N.Y. pp. 559-598.

8.3 Breckinridge, J.C. and M.S. Bergdoll. 1971. Outbreak of food-borne gastroenteritis due to a coagulase-negative enterotoxin-producing Staphylococcus. N. Engl. J. Med. 284: 541-543.

8.4 Genigeorgis, C. 1989. Present state of knowledge on staphylococcal intoxication. Int. J. Food Microbiol. 9:327-360.

8.5 Hirooka, E.Y., E.E. Muller, J.C. Freitas, E. Vicente, Y. Yoshimoto, and M.S. Bergdoll. 1988. Enterotoxigenicity of Staphylococcus intermedius of canine origin. Int. J. Food Microbiol. 7:185-191.

8.6 Jechorek R.P., and R.L. Johnson. 2008. Evaluation of the VIDAS® Staph Enterotoxin II (SET 2) Immunoassay Method for the Detection of Staphylococcal Enterotoxins in Selected Foods: Collaborative Study. J. AOAC Int. 91:164-173.

8.7 Wieneke, A.A., and R.J. Gilbert. 1987. Comparison of four methods for the detection of staphylococcal enterotoxins in foods from outbreaks of food poisoning. Int. J. Food Microbiol. 4: 135-143.