The Eight Hour Reveal Method for Detecting Escherichia Coli O157:h7 in Raw Beef and Environmental Samples.

Laboratory Procedure MFLP-94
September 2000

1. APPLICATION

The method is applicable to the detection of Escherichia coli O157:H7 from raw beef cubes, raw ground beef, and environmental swabs to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act.

2. DESCRIPTION

The method has been shown to produce satisfactory results with artificially contaminated raw cubed beef and raw ground beef as well as environmental swabs from stainless steel in an AOAC study (8.1). < The Microbiological Methods Committee has approved this method for the detection of E. coli O157:H7 in raw beef and environmental swabs only. Other food products should be analysed using other methods available in this Compendium.

3. PRINCIPLE

Following selective enrichment in the Reveal 8 hour broth, a portion of the sample enrichment is placed into the sample port of the Reveal Device initiating flow. The Reveal Device contains antibodies with high specificity to E. coli O157:H7 antigens. These antibodies are bound to colloidal gold and, separately, to a solid support matrix. Any E. coli O157:H7 antigen present will bind to the gold conjugated antibodies forming an antigen - antibody - chromogen complex. This complex flows across a lateral flow membrane and is subsequently bound by antibody immobilized on the membrane. This causes the gold conjugate to precipitate, forming a visible line and indicating a positive reaction. Proper test completion and flow is indicated by a control line which forms further up in the test window and verifies a valid test run. Absence of a control line invalidates the test. Record results at 15 minutes incubation time.

4. DEFINITION OF TERMS

See Appendix A of Volume 3, items 1,2,3,4.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 3.

6. MATERIALS AND SPECIAL EQUIPMENT

1) Reveal Device (Neogen Corporation, phone: 1-800-234-5333, Fax: (517) 372-0108)

2) Reveal 8 hour broth (Neogen Corporation)

3) Biosynth BCM O157:H7+ chromogenic agar (Biosynth, Naperville, IL)

4) Hemorrhagic coli agar

5) MacConkey sorbitol agar (Acumedia, Baltimore, MD)

6) mHC agar or TCCSMAC agar (see MFLP-80)