SOP: Detection of Salmonella in Food Products by the VIDAS Easy Salmonella (SLM) Method

GIFSQ — Global Institute for Food Safety & Quality

Principle

The VIDAS Easy Salmonella (SLM) method is an enzyme immunoassay run on the VIDAS instrument for the detection of Salmonella antigens, using the ELFA technique (Enzyme Linked Fluorescent Assay). The internal surface of the Solid Phase Receptacle (SPR), a pipette-tip-like disposable device, is pre-coated during kit production with anti-Salmonella antibodies, and the reagents for an assay are ready-to-use, pre-dispensed in a sealed multi-chambered strip.

All assay steps are performed automatically and sequentially by the VIDAS instrument. Part of the enrichment broth is inoculated into the reagent strip and cycled in and out of the SPR several times. The antigens present in the sample bind to the anti-Salmonella antibodies coating the interior of the SPR, and unbound components are eliminated during the washing steps. Antibodies conjugated with alkaline phosphatase are then cycled in and out of the SPR and bind to any Salmonella antigens that are themselves bound to the antibodies on 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 as positive or negative.

The method is applicable to the rapid detection of Salmonella spp. to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. Positive results must be confirmed with a cultural method. The method has been validated for use in the food categories dairy products, chocolate and bakery products, fish and seafood, and the food types of heat processed eggs and derivatives, raw processed meat, raw unprocessed meat, and processed cooked multi-ingredient composite foods. Although the method is only approved for these food products, other commodities may be used with it provided they are properly validated following the criteria in the Compendium of Analytical Methods so that the method is fit for purpose for those commodities.

Apparatus

• SX2 broth (ref. 42121, 20 tubes), from bioMerieux Canada, Inc.
• VIDAS family instrument (mini-VIDAS or VIDAS 30)
• VIDAS Salmonella (SLM) kit (ref. 30702): contains the necessary reagents, including standards and controls, for the analysis of 60 test samples
• VIDAS Heat and Go, or a water bath (95–100 °C) or equivalent system
• Buffered Peptone Water (BPW)
• Stomacher and stomacher bags with filter, blender or equivalent
• Pipette calibrated to dispense 500 µL, with disposable tips
• Incubators capable of maintaining 35 °C and 42 °C

Reagents

• Buffered Peptone Water (BPW)
• To prepare: dissolve 10.0 g of peptone, 5.0 g of sodium chloride (NaCl), 3.5 g of disodium phosphate and 1.5 g of potassium dihydrogen phosphate in 1.0 L of distilled water; final pH 7.2 ± 0.2. Dispense into appropriate glassware and autoclave at 121°C for 15 min.
• SX2 broth (kit component, ref. 42121, 20 tubes)

Sample preparation

• Handle the sample units in the laboratory before analysis: except for shelf-stable foods, keep the 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. Analyze the sample units as soon as possible after receipt at the laboratory.
• Preparation for analysis: have sterile BPW available, pre-warmed to 35 ± 1 °C before use. Clean the surface of the working area with a suitable disinfectant.
• 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.
• Each sample unit may be analyzed individually, or the analytical units taken from each sample unit may be combined as appropriate.

Procedure

Aseptically add 25 g (or 25 ml) of sample into a stomacher bag.

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Dilute the sample 1:10 by adding BPW. For composite samples, analytical units may be combined up to 125 g or ml — for example, 125 g or mL of food to 1125 mL of BPW. If alternate analytical units are required, maintain a ratio of 1 part sample material to 9 parts BPW.

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Homogenize the sample using a stomacher.

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Incubate at 35 ± 1 °C for 16–22 hours (non-selective enrichment).

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Allow SX2 broth to come to room temperature before use. After incubation, mix and transfer 0.1 mL of the suspension into 10 mL of SX2 broth.

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Incubate for 22–26 hours at 42 ± 1 °C (selective enrichment).

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If desired, refrigerate the incubated SX2 broth for up to a total of 72 hours (optional; the total refrigeration period used for this step and for the confirmation step must not exceed 72 h).

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Remove the VIDAS Salmonella (SLM) kit from the refrigerator; remove only the reagents needed and let them come to room temperature for at least 30 minutes. Use one “SLM” strip and one “SLM” 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. Components of different lots cannot be mixed, and cannot be used past the expiry date. When a new kit lot is being used, enter the specifications (the factory master calibration curve data) first into the system using the master lot entry card included with each kit; perform the calibration using the standard provided in the kit each time a new lot of reagents is opened, and then every 14 days (refer to the package insert for the master lot data entry and calibration instructions).

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After incubation, homogenize the SX2 broth.

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Prepare the sample for the VIDAS assay by one of two routes:

• Water bath route: transfer an aliquot of SX2 broth into a tube and seal it. 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.
• Heat and Go route: transfer 0.5 mL of the SX2 broth into the sample well on the strip. Label the strip correctly and heat for 15 ± 1 minutes (refer to the user’s manual). Remove the strip and leave it to cool for 10 minutes. Do not heat the standard and controls.

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Initiate the assay as directed in the VIDAS operator’s manual. Insert the SPR and strips into the positions that correspond to the VIDAS section indicated by the work list. Check that the colour labels with the assay code on the SPR and the reagent strips match.

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The instrument analyzes the results automatically. Immunoassays producing relative fluorescence values (RFV) of 0.23 indicate the presumptive presence of Salmonella in the test sample.

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Using the non-boiled SX2 broth, or the refrigerated non-boiled SX2 broth, proceed with the plating and confirmation steps of a cultural method — the GIFSQ SOP for Isolation and Identification of Salmonella from Food and Environmental Surface Samples (MFHPB-20).

Precautions

• 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”.
• If the analyst uses any variations of the media listed here (either a commercially available product or media made from scratch), it is the responsibility of the analyst or the Laboratory Supervisor to ensure equivalency.
• 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 the 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.

References

• Health Products and Food Branch, Health Canada. Detection of Salmonella in food products by the VIDAS Easy Salmonella (SLM) method (Laboratory Procedure MFLP-40, November 2011). Compendium of Analytical Methods, Volume 3.
• ISO/IEC 17025:2005. General requirements for the competence of testing and calibration laboratories.
• Reid, A. 2009. Isolation and identification of Salmonella from foods and environmental samples (MFHPB-20). The Compendium of Analytical Methods. In: Volume 2. The Compendium of Analytical Methods.