The Dupont™ Lateral Flow System™ Method for Detecting E. coli O157 in Raw Ground and Raw Boneless Beef

Laboratory Procedure MFLP-19
March 2006

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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
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 Escherichia coli O157 in raw ground beef and raw boneless beef in support of compliance activity relative to Sections 4 and 7 of the Food and Drugs Act.

2. DESCRIPTION

The DuPont™ Lateral Flow System™ (LFS) E. coli O157 test has been designed to detect E. coli O157 in raw ground beef and raw boneless beef . The test kit permits presumptive detection and identification of the target pathogen when present at levels of one E. coli O157 organism per 25 grams of sample (0.04 cells/g). The test provides a simplified process with satisfactory results in 8 and 18 hours (using DuPont™ LFS media for E. coli O157) or 20 hours (using standard enrichment media).

3. PRINCIPLE

The Dupont™ LFS immunoassay test uses a double antibody "sandwich" format. An antibody that is specific to E. coli O157 is sprayed and immobilized in a test line on the surface of a membrane. A second antibody reagent, also recognizing E. coli O157 and labelled with colloidal gold, is contained within a reagent pad upstream from the test line on the membrane. As the liquid sample moves by capillary action through the reagent pad, the antibody-gold reagent specifically binds to E. coli O157 and moves with the sample into the test membrane. Moving up the test membrane, the sample passes through the test line, where the immobilized E. coli O157 antibody captures the E. coli O157-antibody-gold complex, forming an antibody-E. coli O157 "sandwich." The test line then develops a red color. In the absence of E. coli O157, no antibody-E. coli O157 sandwich is formed, and the test line does not turn red. Reagents immobilized at the control line captures 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. Therefore, a single (control) line on the membrane indicates a negative sample, and two (sample and control) lines indicate a presumptive positive sample.

4. DEFINITION OF TERMS

See Appendix A of Volume 3.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 3.

6. MATERIALS AND SPECIAL EQUIPMENT

6.1 8 or 18 Hour Test

1. DuPont™ LFS E. coli O157 Test Kit (PN 18220014 - 50 test strips)
2. DuPont™ LFS E. coli O157 Media (PN 18230016 - 500 grams)
3. Control cultures, refer to MFLP-80
4. Balance with a range of 25 to 1000 grams
5. Stomacher with filtered bags (Seward Stomacher® 400 Circulator or equivalent)
6. Incubator capable of maintaining 42°C with agitation (agitation is optional)
7. Test tubes (12 x 75 mm) with rack
8. Pipettes (400 µL) for transfers

6.2 20 Hour Test

1. Requires all of the materials and special equipment listed in 6.1, except # 2) and # 6)
2. Modified EC with novobiocin (m EC+ n) (see MFLP-80 (8.1))
3. Incubator capable of maintaining 37°C

7. PROCEDURE

7.1 E. coli O157 Test Protocol (8 or 18 hour)

7.1.1 Preparation of the Dupont™ LFS Enrichment broth.

7.1.1.1 Sterilize one liter of water either by autoclaving or filtering (pore size 0.2 µm) into a sterile container. Equilibrate to 42°C in a water bath or incubator.

7.1.1.2 Add 25.2 grams of DuPont™ LFS E. coli O157 Media to the sterilized water. Shake vigorously until the media is dissolved.

7.1.2 Enrichment of Samples

7.1.2.1 Prewarm prepared enrichment broth to 42°C.

7.1.2.2 Prepare a 1:10 dilution of the food by aseptically adding 25 g (or other suitable analytical unit, such as 65 g for ground beef) into 225 mL (or amount necessary to maintain the 1:10 ratio) of the enrichment broth and stomach.

7.1.2.3 Close the bag loosely and incubate 8 hours at 42°C with or without agitation, or up to 18 hours at 42°C without agitation.

7.1.2.4 After at least 8 hours, remove the bag from the incubator and use a gentle swirling motion to mix the contents.

7.1.2.5 Optional Heat-Kill Step: The DuPont™ LFS E. coli O157 test has been developed to detect both live and heat-killed organisms. To detect heat-killed organisms after incubation, use a sterile pipette to transfer 5 mL enriched sample to a glass tube. Heat in boiling water for 10 minutes, then allow tube to cool to room temperature before proceeding with test.

7.1.3 Test Samples.

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

7.1.3.2 Transfer 400 µL of enriched sample to a tube

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

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

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

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

7.1.3.7 If at 10 minutes the test strip only shows a clearly visible control line, then the sample is negative for E. coli O157.

7.1.3.8 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 E. coli O157.

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

7.1.4 Confirmation of Presumptive Positive Results.

Use enriched samples to confirm presumptive positives as described in MFLP-80 (8.1)

7.2 E. coli O157 Test Protocol (20-hour)

7.2.1 Preparation of m EC+ n Broth

7.2.1.1 Add 36.1 g of modified EC media to 1 liter of distilled water. Shake vigorously until dissolved, and adjust the pH to 6.9, if necessary. Autoclave at 121°C for 15 minutes.

7.2.1.2 Allow the broth to cool, then add 5 mL sterilized novobiocin solution (4 mg/mL active sodium novobiocin). Final novobiocin concentration is 20 mg/liter. Equilibrate the broth to 37°C.

7.2.2 Enrichment of Samples.

7.2.2.1 Prewarm prepared enrichment broth to 37°C.

7.2.2.2 Prepare a 1:10 dilution of the food by aseptically adding 25 g (or other suitable analytical unit, such as 65 g for ground beef) into 225 mL (or amount necessary to maintain the 1:10 ratio) of the enrichment broth, and stomach.

7.2.2.3 Close the bag loosely and incubate 20 hours at 37°C.

7.2.2.4 After 20 hours, remove the bag from the incubator and use a gentle swirling motion to mix the contents.

7.2.2.5 Optional Heat-Kill Step: Use a sterile pipette to transfer 5 mL enriched sample to a glass tube. Heat in boiling water for 10 minutes, then allow tube to cool to room temperature before proceeding with test.

7.2.2.6 Continue test as described in 7.1.3.

7.2.3 Confirmation of Presumptive Positive Results

Use enriched samples to confirm presumptive positives as described in MFLP-80 (8.1)

8. REFERENCES

8.1 Warburton, D., and D. Christensen. 2006. Isolation of E. coli O157 in Foods (MFLP-80). Volume 3, Compendium of Analytical Methods, Health Canada, Ottawa. Published on the Food Directorate's (Health Canada) website at http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php.