SOP: Detection of Staphylococcal Enterotoxins in Foods

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This SOP detects staphylococcal enterotoxins — the pre-formed toxins that cause staphylococcal food poisoning — in foods. The toxins are extracted from the food by acidified aqueous extraction; the extract is concentrated by dialysis against polyethylene glycol; and the concentrated extract is tested with commercial immunoassays that detect staphylococcal enterotoxins SEA through SEE. Polyvalent enzyme-linked immunoassays (ELISA) or enzyme-linked fluorescent immunoassays (EFLA) detect SEA–SEE together; monovalent assays distinguish the individual types. For meat and poultry products, the SOP also gives the FSIS two-kit procedure: screening with VIDAS SET2 (an enzyme-linked fluorescent assay) and confirmation of non-negative samples with RIDASCREEN SET Total (an enzyme-linked immunosorbent assay).

2. Apparatus required

• Blender or homogenizer
• Analytical balance and weigh boats (balance sensitivity 0.1 g)
• pH meter, for extraction pH and buffer pH (adjustments within ± 0.1 pH unit)
• Refrigerated centrifuge, 2–8 °C
• Temperature-controlled room or refrigerator, 2–8 °C
• Incubator, 35–37 °C
• Biological safety cabinet
• Glass or polypropylene laboratory-ware (to avoid toxin adsorption)
• Dialysis membrane, MWCO 6,000–8,000 Daltons, flat width 23 ± 2 mm, with closures
• Tray for dialysis
• Separatory funnel
• Filter cloth, for collecting debris after centrifugation (several layers of pre-wetted coarse cheesecloth are commonly used)
• Vacuum conical tube filtration devices with 0.22 µm membrane, for filtering liquid culture supernatants
• Orbital shaker
• Micropipettes capable of dispensing 100 µl, 200 µl and 500 µl
• Sterile stomacher bags (sized to hold the 1:10 diluted sample) or Whirl-Pak bags
• Disposable polypropylene screening columns
• Microplate reader, 450/620 ± 10 nm

3. Reagents

• A. Phosphate buffered saline (PBS) solution, pH 7.3 + 0.2 (NaCl/Na₂HPO₄: 145 mM/10 mM)
• To prepare: dissolve 9 g of NaCl and 3.58 g of Na₂HPO₄ in 1 L of distilled water; adjust the pH to 7.2 ± 0.2 using HCl
• B. 30% (w/v) polyethylene glycol (PEG) solution, PEG mol wt 20,000
• To prepare: add 30 g of PEG for each 70 mL of distilled water
• C. 1 N (or 0.1 N) NaOH
• D. 1 N (or 0.1 N) HCl
• E. Buffered peptone water (BPW), as the FSIS sample diluent and additional negative control
• F. 0.5% sodium hypochlorite solution, made fresh daily, for decontaminating work surfaces (a 2% solution may be used up to 30 days after preparation)
• G. Nutrient broth (tryptic soy broth or brain heart infusion broth), for pre-enrichment of staphylococcal cultures
• H. Commercial enterotoxin detection kits, used according to the kit instructions: VIDAS SET2, Ref. 30705 (an automated polyvalent enzyme-linked fluorescent assay detecting SEA–SEE) and RIDASCREEN SET Total, R4105/R4106 (a manual polyvalent enzyme-linked immunoassay detecting SEA–SEE)
• The kit must be able to detect enterotoxin at a minimum level of 0.5 ng/g, with relative sensitivity above 90% and relative specificity above 90%
• Kit manufacturers may require different buffers or solvents; follow the kit’s own buffer requirements

4. Sample preparation

• If possible, grind the entire food product, or portions from representative selections of the product, in a blender to homogenize the sample so that any enterotoxin present is evenly distributed.
• For cheeses with rind, take the cheese sample as 10% rind and 90% cheese.
• For dried products, use equal amounts of water with the product to blend.

5. Procedure

5a. Extraction of enterotoxin from the food portion (FDA BAM)

Perform this procedure and any other procedure that may generate aerosols of pathogenic microorganisms or enterotoxins in an approved biological safety cabinet.

Prepare the dialysis membrane first: cut the membrane long enough to hold the extract to be concentrated
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Soak the tubing in 2 changes of distilled water to remove the glycerol coating
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Tie one end of the tubing with a membrane closure, or with 2 knots close together
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Fill the tube with distilled water and test for leaks by squeezing the filled sac while holding the open end tightly closed
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Empty the sac and keep it in distilled water until use
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Weigh 25 g of sample in a glass beaker and transfer the test portion to a blender with 40 mL of distilled water; blend at high speed for 3 minutes to make a homogenized slurry (do not add water to liquid samples; proceed to the shaking step)
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Allow the toxin to diffuse by shaking the sample at room temperature for 30 minutes
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Acidify the mixture with 0.1 N HCl to a pH between 3.5 and 4.0 (if the pH drops below 3.0, prepare another 25 g portion)
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Transfer the acidified slurry to 50 mL conical propylene tubes and centrifuge at 3,130 × g for 20 minutes at 5 °C (lower speeds with longer centrifuging time can be used, but clearing of some foods is less effective; separation of fatty material requires refrigerated centrifuging)
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Decant the supernatant fluid into an 800 mL beaker through cheesecloth or other suitable filtering material placed in a separatory funnel (if the supernatant is not clear, centrifuge again and decant through the cheesecloth)
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Test the pH, which should be between 3.5 and 4.5; if the pH is correct, neutralize the mixture with 0.1 N NaOH to a pH between 7.4 and 7.6
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If the pH is above 4.5, repeat the acidification; if the pH is below 3.0 or above 9.0, repeat the process with another 25 g food portion
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If enough sample remains for repeat testing, an aliquot can be removed for screening with a validated assay; if the screening result is negative, the remaining extract must be concentrated by dialysis and retested

5b. Dialysis concentration of the extract (FDA BAM)

Place the extract in the prepared dialysis sac
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Lay the closed sac in a tray and immerse it in 30% (w/v) PEG
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Hold at 5 °C until the volume is reduced to 15–20 mL or less (this may take 24–72 hours; if the volume has not reduced after holding overnight, add powdered PEG to the tray)
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Remove the sac from the PEG and wash the outside thoroughly with water to remove adhering PEG; soak in distilled water for 1–2 minutes
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Pour the contents into a small beaker
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Rinse the inside of the sac with 2–3 mL of distilled water (use PBS for milk and dairy products) by running fingers up and down the outside of the sac to remove material from the sides of the tubing; repeat rinsing until the rinsate is clear, keeping the volume as small as possible
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Adjust the pH of the extract to 7.4–7.6
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Analyze the concentrated extract within 48 hours, storing it at 3–5 °C; otherwise freeze the extract at −18 to −20 °C (the method prints 18–20 °C; the minus sign is missing in the print) and thaw it completely at 3–5 °C before testing (some assays, such as VIDAS SET2, require immediate analysis)

5c. Testing the extract with the immunoassay kits (FDA BAM)

Test the extract with the chosen validated immunoassay kit according to the kit instructions
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Dialysis concentration improves method sensitivity and selectivity; preliminary testing without concentration is acceptable only where time constraints require it
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Perform dialysis concentration on all dairy products before analysis
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Check for interferences from endogenous enzymes: products containing endogenous lactoperoxidase or alkaline phosphatase can give non-specific reactions with kits that use those enzymes for detection (such as VIDAS SET2, which uses alkaline phosphatase)
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Confirm every positive result with an alternative method that uses a different detection enzyme (for example, RIDASCREEN SET Total, which uses lactoperoxidase, as the alternative to VIDAS SET2)
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To remove endogenous alkaline phosphatase before repeating VIDAS SET2: transfer 600 µL of the concentrated extract to a tube, heat at 80 °C for 2 minutes, and repeat the assay with the cooled concentrate (some loss of enterotoxin may occur)
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To check for intrinsic lactoperoxidase before using a lactoperoxidase-based kit: transfer 100 µL of concentrated extract to a tube, add 50 µL each of the kit’s substrate and chromogen solutions, and mix; a blue-green color shows intrinsic lactoperoxidase is present and interfering, so a different detection method must be used

5d. Testing enterotoxins from bacterial cultures (FDA BAM)

Staphylococcal strains suspected of producing enterotoxins can be pre-enriched in nutrient broth.

Transfer two or three morphologically similar colonies to 10 mL of nutrient broth
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Incubate at 35–37 °C overnight on an orbital shaker
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Centrifuge the cultures for 5 minutes at 3,500 × g at 10 °C
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Filter the supernatant with a 0.22 µm vacuum filter device or other closed system, in a biological safety cabinet, to avoid aerosolizing the enterotoxins
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Dilute culture supernatants with PBS if the enterotoxin level is outside the linear range of the kit

5e. Variant — FSIS procedure for meat and poultry products (USDA FSIS MLG 39.03)

This variant screens and confirms staphylococcal enterotoxins A, B, C, D and E in FSIS regulated products: ready-to-eat products, pasteurized egg products and raw meat products. All measurements carry a tolerance range of ± 2% unless stated otherwise.

Prepare the sample as for the Salmonella method (MLG 4), keeping the matrix-to-medium ratio; the sample portion may be reduced as long as the ratio is maintained
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Aseptically transfer an adequate volume for the procedures below, following any additional sample preparation in the kit instructions
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Let the sample sit for 15 minutes at 18–25 °C
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Centrifuge at 3,000–5,000 × g at 18–25 °C for 15 minutes
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Filter the supernatant through a screening column
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Check the filtrate and adjust the pH to 7.5–8.0 with 1 N NaOH if necessary
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Include the method controls with each run: for VIDAS SET2, up to 5 controls per run — kit positive control (C1) in one reagent strip, kit negative control (C2) in one reagent strip, BPW as an additional negative control in one reagent strip, and S1 run twice on 2 strips if required (every 14 days); for RIDASCREEN SET Total, 3 controls per run — kit positive control 100 µL undiluted, kit negative control 100 µL undiluted, and BPW as an additional negative control
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Run VIDAS SET2 according to the kit instructions; the kit’s software reads and interprets the results automatically (the validated sensitivity is below 1 ng of enterotoxin per gram or mL of sample)
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If a sample is negative on VIDAS SET2, no further analysis is needed; if a sample cannot be ruled out, test it with RIDASCREEN SET Total according to the kit instructions (the validated sensitivity is below 1 ng of enterotoxin per gram or mL of sample; the substrate/chromogen incubation may take up to 25 minutes to bring the positive control to at least 1.0)
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Read the RIDASCREEN results at 450/620 ± 10 nm with the microplate reader

6. Reading and reporting results

For VIDAS SET2, a test is valid when the negative control is ≤ 0.13, the positive control is ≥ 0.13 and the standards are within the instrument’s relative fluorescence value (RFV); if not, recalibrate the instrument. A sample is negative when the test is valid and the sample value is below 0.13; a sample is positive when the test is valid and the sample value is ≥ 0.13.

For RIDASCREEN SET Total, the run is valid when the negative control is below 0.1 and the positive control is above 1.0. The cut-off value is the OD value of the negative control plus 0.15. A sample is POSITIVE when the test is valid and the sample absorbance is at or above the cut-off; a sample is NEGATIVE when the sample absorbance is below the cut-off.

Interpret the two-kit combination as follows: VIDAS SET2 negative — Negative. VIDAS SET2 positive and RIDASCREEN SET Total negative — Negative. VIDAS SET2 positive and RIDASCREEN SET Total positive — Confirmed Positive for staphylococcal enterotoxin.

Positive results are reported as staphylococcal enterotoxin detected in × g (mL) of product; negative results as staphylococcal enterotoxin not detected in × g (mL) of product.

The presence of staphylococcal enterotoxin in a food product is reported to the Federal Select Agent Program by completing APHIS/CDC Form 4.

7. Precautions

• Staphylococcal enterotoxins are highly toxic: perform all procedures that may create aerosols in an approved biological safety cabinet.
• Staphylococcal enterotoxins (SEA, SEB, SEC, SED and SEE) are select agents.
• Staphylococcal enterotoxins are heat stable; they are not denatured except at high temperature for long periods (autoclave at 121 °C at 15 PSI for 60 minutes).
• Wear laboratory coat, gloves and protective eyewear; work under Biosafety Level 2; only trained personnel perform these tests.
• Decontaminate work surfaces before and after use with 0.5% sodium hypochlorite made fresh daily, or 2% sodium hypochlorite used within 30 days of preparation; place contaminated non-disposable materials in decontamination solution until autoclaved, and disposable materials in closed biohazard bags autoclaved at least 60 minutes at 121 °C and 15 psi before disposal.
• Keep the pH during extraction and the pH of extraction buffers within ± 0.1 pH unit of the stated value.
• Use glass or polypropylene laboratory-ware so toxins do not adsorb to surfaces.
• Filtrate supernatants through 0.22 µm closed filter devices to avoid aerosolizing enterotoxins.
• Analyze dialysis-concentrated extracts within 48 hours (stored at 3–5 °C); VIDAS SET2 requires immediate analysis of the concentrate.
• Watch for endogenous enzyme interference (lactoperoxidase, alkaline phosphatase) and confirm every positive with an alternative method using a different detection enzyme.

8. References

• FDA, Bacteriological Analytical Manual, Chapter 13B: Staphylococcal Enterotoxins Detection Methods, September 2022 Edition.
• USDA Food Safety and Inspection Service, Laboratory Guidebook, MLG 39.03: Preliminary and Confirmatory Testing of FSIS Regulated Products for Staphylococcal Enterotoxins, effective 04/01/18.