Identification of Presumptive Positive Verocytotoxigenic Escherichia Coli by the Polymerase Chain Reaction

Laboratory Procedure MFLP-86
January 2003

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Kim Ziebell
Laboratory for Foodborne Zoonoses
Health Canada,
Guelph, Ontario N1G 3W4
E-mail: Kim_Ziebell@hc-sc.gc.ca

1. APPLICATION

The method is applicable to the rapid identification of Verocytotoxigenic Escherichia coli (VTEC) isolated from foods and other samples using APHD Methodology (6.1), HPFB methodology (6.3) and other methodology to detect Escherichia coli O157 and/or other VTEC. It can be applied to the definitive identification of presumptive positive colonies isolated on MacConkey agar, and/or any suitable agar medium routinely used in the isolation of this pathogen from enrichment cultures . When product-based compliance action is anticipated, and where stipulated, HPFB Methodology should be used exclusively or for further confirmation of polymerase chain reaction-positive colonies.

2. PRINCIPLE

Following the detection procedure, a portion of presumptive positive colonies plated on MacConkey Agar (Mac) is subjected to a polymerase chain reaction (PCR) procedure* which amplifies a specific DNA sequence of the toxin gene. The priming oligonucleotides (primers) used in the PCR are highly specific for VTEC, and do not amplify DNA present in any other non-VTEC organisms. The resulting amplified DNA fragment has a specific molecular size, defined by the primers, and is readily identified by agarose gel electrophoresis. The entire procedure identifies presumptive positive colonies within 5 h, and can replace the usual screening and confirmation tests thus providing considerable savings on time, labour and cost of the analysis. The PCR technique has proven to be a highly specific and sensitive method for the identification of VTEC from a variety of samples.

*The polymerase chain reaction (PCR) process is covered by U.S. patents owned by Hoffman-LaRoche.

3. MATERIALS AND SPECIAL EQUIPMENT

1. Thermal cycler ( Model 9600, Perkin-Elmer Cetus) or equivalent.
   
   
2. Microwave oven or hot plate.
   
   
3. Submarine gel casting tray, buffer reservoir and power pack.
   
   
4. Shortwave UV light table (transilluminator) to visualize stained DNA in agarose gels.
   
   
5. Photodocumentation system (optional, for photographic records), including polaroid camera (hand-held or fixed)
   
   
6. Pipettors: to cover range of volumes (0.5-1000/ L).
   
   
7. sterile Milli-que (MQ) water or equivalent (purified or distilled)