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Applied and Environmental Microbiology, November 1999, p. 4775-4780, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Application of a DNA Hybridization-Hydrophobic-Grid Membrane Filter Method for Detection and Isolation of Verotoxigenic Escherichia coli

E. C. D. Todd,1,* R. A. Szabo,1 J. M. MacKenzie,1,dagger A. Martin,2 K. Rahn,3 C. Gyles,4 A. Gao,4 D. Alves,5 and A. J. Yee6

Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health Canada, Ottawa, Ontario K1A 0L2,1 Education, Research and Laboratories Division, Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Ontario N1G 4Y2,2 Guelph Laboratory, Health Protection Branch, Health Canada, Guelph, Ontario, N1G 3W4,3 Department of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1,4 Livestock Technology Health Management, Ontario Ministry of Agriculture, Food and Rural Affairs, Fergus, Ontario N1M 2W3,5 and Laboratory Services Division, University of Guelph, Guelph, Ontario N1H 8J7,6 Canada

Received 12 April 1999/Accepted 4 August 1999

Verotoxigenic Escherichia coli (VTEC) strains were isolated from food and animal fecal samples by using PCR to screen for the presence of VTEC after broth enrichment and then filtering VTEC-positive cultures through hydrophobic-grid membrane filters (HGMFs) which were incubated on MacConkey agar. The filters were probed with a digoxigenin-labeled PCR product generated by amplification of a conserved verotoxin gene sequence. Replication of the growth on filters allowed probe-positive colonies to be picked. When ground beef samples were inoculated with VTEC strains, 100% of the strains were recovered, and the detection limit was 0.1 CFU per g. Similar results were obtained with seven types of artificially contaminated vegetables. A survey of 32 packages of vegetables and 23 samples of apple cider obtained at the retail level did not reveal the presence of VTEC. However, the intestinal fecal contents of a moose, 1 of 35 wild mammals and birds examined, contained E. coli O157:H7. The DNA hybridization-HGMF method was also used in a prevalence survey of 327 raw and 744 ready-to-eat products; VTEC strains were recovered from 4.9% of the raw products and 0.7% of the ready-to-eat products. No serotype O157:H7 strains were detected. This method is particularly suited for surveys in which low numbers of VTEC-positive samples are expected and isolates are required.

* Corresponding author. Mailing address: Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Sir Frederick G. Banting Research Centre, Building Locator 2204A2, Ottawa, Ontario K1A 0L2, Canada. Phone: (613) 957-0887. Fax: (613) 941-0280: E-mail: ewen_todd{at}hc-sc.gc.ca.

dagger Deceased.

Applied and Environmental Microbiology, November 1999, p. 4775-4780, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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