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Detection of Verotoxins VT 1 And VT 2 by The Merck Duopath® Verotoxin Kit

Laboratory Procedure MFLP-83
December 2006

(PDF Version - 26 K)

Don Warburton
and the Microbiological Methods Committee
Evaluation Division
Bureau of Microbial Hazards,
Food Directorate, 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 verotoxins produced by Escherichia coli O157 in foods to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and Guideline 10 (9.2 ).

2. Principle

The Duopath® Verotoxins Gold Labelled Immuno Sorbent Assay (GLISA) test is an immunochromatographic rapid test intended to be used in food laboratories for the qualitative detection of Verotoxins (Shiga-like toxins) 1 and 2 from Verotoxinogenic E. coli O157:H7 isolated from food. This test has been validated and received AOAC approval (AOAC license no 020402) for detection of Verotoxins 1 and 2 from isolates of Verotoxin-producing E. coli (including E. coli O157:H7).

Among the E. coli human pathogens, verotoxin (Shiga-like toxin) forming strains (VTEC) have gained in importance in recent years. The group of enterohaemorrhagic E. coli (EHEC) with its highly pathogenic serovars O157:H7, O26, O103, O111, O145, and other strains are of particular concern. Production of verotoxins is a common characteristic of this group of bacteria. Verotoxins can be classified into two main categories, verotoxin 1 (VT1, SLT1, Stx1) and verotoxin 2 (VT2, SLT2, Stx2). EHEC strains usually produce either VT1 or VT2, or both. EHEC are capable of initiating life-threatening illnesses, particularly in those with immune deficiency, young children and the elderly. The main sources of infection are contaminated, raw or insufficiently heated foods of animal origin, e.g. meat and dairy products. Other foods such as unpasteurized apple juice, fresh fruits, vegetables, as well as other foods have been implicated. The reservoir for EHEC is the feces of the cattle, sheep and goats. The incidences of food infection caused by E. coli O157 demands reliable and rapid methods of detection.

3. Definition of Terms

See Appendix A of Volume 3.

4. Collection of Samples

See Appendix B of Volume 3.

5. Materials and Special Equipment

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

1. Media, including broths and agars, and materials specified in MFLP-80 (or equivalent)
2. CAYE Broth, mod. acc. to Evans, and CAYE Broth Supplement C (Merck)
3. Polymyxin-Solution: Polymyxin-B-sulfate (Bacillus Cereus selective supplement) (Merck)
4. Incubators capable of maintaining 35 and 42°C
5. Control cultures. In conjunction with the information provided in MFLP-80, use cultures that produce VT1 and /or VT 2. In addition, use negative controls that do not produce VT

6. Procedure

6.1 Handling of Sample Units

Refer to MFLP-80

6.2 Preparation for Analysis

Refer to MFLP-80 (or other compendial methods for E. coli O157). Refer to specifics of alternate methods for variations in media, temperature of incubation etc.

6.3 Preparation of Sample

Refer to MFLP-80.

6.4 Identification of VT 1 and/or VT2 from E. coli O157:H7 or E. coli O157:NM

6.4.1 Follow MFLP-80 (or equivalent methodology) to detect viable E. coli O157 colonies.

6.4.2 Pick 1-5 confirmed E. coli O157 colonies from the CT-SMAC or TCCSMAC (or other agars listed in MFLP-80) and inoculate 1 mL of the CAYE broth containing CAYE broth supplement C. Incubate for 6-24 h at 35-37°C

6.4.3 Pipette 180 μL CAYE-culture into a small sterile test tube.

6.4.4 Add 20 μL Polymyxin B solution and mix. Incubate the culture sample for 10 min. at 35-37°C.

6.4.5 Allow to cool to room temperature.

6.4.6 Prior to use allow the polymyxin B culture sample and the required number of foil sealed test devices to reach room temperature.

6.4.7 Remove the foil pouches from the required number of Duopath® Verotoxins devices and place on a flat surface. Perform the test within 2 hours of opening.

6.4.8 Gently swirl the polymyxin B culture sample to mix.

6.4.9 Using a micropipetter and disposable pipette tip, draw up 150 μL and dispense it into the circular sample port on the test device. Alternatively use a disposable transfer pipette and dispense five (5) free falling drops into the circular sample port on the test device (about 150 μL).

6.4.10 Incubate at room temperature and read within 20 min.

7. Interpretation

7.1 The test is working correctly if a distinct red line appears in the control zone (C) within the read time of 10-20 minutes. If the control zone (C) line does not appear, discard the test and repeat with a new test kit.

7.2 A sample can be considered positive if at or prior to the read time, red lines appear in the test (VT1 and/or VT2) zone and control (C) zone.

7.3 A sample can be considered negative if no red line appears in the test (VT1 and VT2) zone but does appear distinctly in the control (C) zone within 10-20 minutes after the application of the sample to the device.

7.4 The result must be red to be considered positive. A dark line that is not red should be considered negative.

7.5 A positive result indicates that Verotoxin 1 and/or 2 (Shiga-like toxins) from E. coli were detected in the sample. A negative result indicates that neither Verotoxin 1 nor 2 were detected in the sample.

8. Reporting

Colonies exhibiting characteristic E. coli O157 morphology on selective agar, that have been biochemically and serologically confirmed as outlined in MFLP-80 and that exhibit production of VT1 and/or VT2, are reported as verotoxic E. coli O157 or E. coli O157:NM (as appropriate). If the presence of H7 flagella have been confirmed (an optional step of MFLP-80), report the presence of verotoxic E. coli O157:H7.

9. References

9.1 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.sc-hc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php.

9.2 Guideline No 10. Health Canada (HC) website: http://www.hc-sc.gc.ca/fn-an/legislation/guide-ld/guidelines_raw_ground_beef-directives_boeuf_hache_cru-eng.php.