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The 48 H Dupont™ Lateral Flow System™ Method for the Detection of Salmonella Species from Raw and Processed Meats

Laboratory Procedure MFLP-18
November 2007

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Health Products and Food Branch
Ottawa

Microbiological Methods Committee
Microbiology Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, 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 species in raw (ground chicken, turkey, beef and pork, as well as chicken carcass wash) and processed meats (turkey deli meat only).

Note: While this method is only approved for certain food products, as listed above, it is assumed that this method can be used with other commodities. It is imperative that the Laboratory Supervisor ensures that proper validation is completed for other commodities to fulfill SCC Acreditation requirements. It is requested that these validation data be forwarded to Don Warburton (address above) so the "Application Section" can be expanded to include these new commodities

2. Description

2.1 DuPont™ Lateral Flow System™ Salmonella tests have been designed to detect Salmonella species in raw and processed meats. The test permits presumptive detection of the target pathogen when present at levels of one Salmonella organism per 25 grams of sample. The test provides a simplified process with presumptive results in 24 hours (using DuPont™ Lateral Flow System™ media for Salmonella) or 48 hours (using standard enrichment media). Salmonella test strips are designed to be used by trained technicians who follow good microbiology laboratory practices. The protocol involves the use of potentially hazardous microorganisms so appropriate safety practices must be observed.

It is recommended that the analyst read the Limitation of Warranty and Liability (in the product insert) before using product.

3. Principle

3.1 This immunoassay test uses a double antibody sandwich format. An antibody that is specific to Salmonella is sprayed and immobilized in a test line on the surface of a membrane. A second antibody reagent, also recognizing Salmonella and labeled with colloidal gold, is contained within a reagent pad upstream from the test line on the membrane.

3.2 As liquid sample moves by capillary action through the reagent pad, the antibody-gold reagent specifically binds to Salmonella and moves with the sample into the test membrane. Moving up the test membrane, the sample passes through the test line, where the immobilized Salmonella antibody captures the Salmonella-antibody-gold complex, forming an antibody-Salmonella "sandwich." The test line then develops a red color. In the absence of Salmonella, no antibody-Salmonella sandwich is formed, and the test line does not turn red.

3.3 Reagents immobilized at the control line capture excess gold reagent passing through the test line. This causes the control line to develop a red color, which indicates that the test flowed correctly on the strip.

3.4 Therefore, a single (control) line on the membrane indicates a negative sample, and two (sample and control) lines indicate a positive sample.

4. Definition of Terms

See Appendix A of Volume 3.

5. Collection of Samples

See Appendix B of Volume 3.

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6. Materials and Special Equipment

Note: 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".

The media and reagents listed below are commercially available and are to be used, prepared and/or sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 for the formula of most of the individual media.

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

1) DuPont™ Lateral Flow System™ Salmonella Test Kit (PN 18220009 - 50 test strips)

2) Buffered Peptone Water (BPW) - commercially available

3) Lactose Broth (LB) -commercially available

4) TT (Tetrathionate) Broth Base, Hajna - commercially available

5) Balance - 25 to 1000 grams with 0.2 g sensitivity

6) Stomacher with filtered bags (Seward Stomacher® 400 Circulator or equivalent)

7) Incubators capable of maintaining 35 and 42 +/- 0.5°C

Note: It is the responsibility of each laboratory to ensure that the temperature 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 35 +/-1.0°C. Similarly, lower temperatures of 30 or 25°C 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 higher temperatures on the microorganism being isolated.

7) Test tubes (15 ml) with rack

8) Plastic tubes (12 x 75 mm) with rack

9) Pipettes (400 µL) for transfers

10) positive and negative controls (use ATCC cultures or equivalent)

7. Handling of Sample Units

7.1 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.

7.2 Thaw frozen samples at room temperature within 60 min; if this is not possible, thaw the samples at refrigerator (4 to 10°C) temperature.

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

7.4 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.

7.5 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.

8. Sample Preparation and Enrichment

8.1 Compositing of Analytical Units

8.1.1 To reduce the workload, up to 15 x 25 g (mL) analytical units may be composited into a single test sample (e.g. 375 g or mL). When compositing maintain the 1:10 ratio of sample to primary enrichment broth.

8.1.2 If a sample unit consists of more than one container, aseptically mix the contents of the containers prior to withdrawal of the analytical unit. If not possible or practical, the analytical unit shall then consist of equal portions from each of the containers.

8.2 Primary enrichment

8.2.1 Add 25 grams of sample to a sterile Stomacher bag.

8.2.2 Add 225 mL of Buffered Peptone Water to the bag. (Or Lactose Broth for processed meats like Turkey Deli Meat) to the bag.

8.2.3 Blend or stomach samples for 1-2 minutes.

8.2.4 A positive Salmonella and a negative medium control should be set up in parallel with the test samples.

8.2.5 Incubate the pre-enrichment mixture and the positive and negative controls at 35°C for 22-26 hrs.

8.2.6 Remove the bag from the incubator and gently mix contents using a gentle swirling motion.

Note: The negative medium control should not show any evidence of growth after incubation whereas the absence of growth in the positive control would invalidate test results.

8.3 Secondary enrichment

8.3.1 From BPW enrichment broth, transfer 1 mL to 10 mL of TT (Hajna) broth. Incubate for 22-26 hours at 42°C.

8.4 Test samples

8.4.1 Arrange and mark tubes for each sample in a rack.

8.4.2 Transfer 400 µl of enriched sample to a tube.

8.4.3 Remove the required number of test strips from the canister. Do not remove the label on the strip.

8.4.4 Insert the strip with arrows facing downwards into the tube.

8.4.5 Allow the strip to develop for 10 minutes, then check the results as follows:

8.4.6 At least one line, the control line, should always develop. A red line in this position indicates that the strip is functioning properly.

8.4.7 If at 10 minutes the test strip only shows a clearly visible control line, then the sample is negative for Salmonella.

8.4.8 A red line appearing below the control line is the test line and indicates a positive result. If the test strip displays two (2) red lines, the test is complete and the sample is positive for Salmonella.

8.4.9 If no control line develops within 10 minutes, the test is invalid and needs to be repeated.

Note: Test strip results should always be interpreted after 10 minutes. Test strips interpreted after 20 minutes are invalid.

8.5 Confirmmation of Presumptive Positive Results

8.5.1 Positive LFS results must be confirmed culturally as described in MFHPB-20, using the secondary enrichment

9. References

9.1 D'Aoust. J-Y, and U. Purvis. 1998. MFHPB-20. Isolation and Identification of Salmonella from Foods. In: Volume 2: The Compendium of Analytical Methods. Health Protection Branch, Ottawa, Canada. (http://www.hc-sc.gc.ca/food-aliment/mh-dm/mhe-dme/compendium/e_index.html)