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Applied and Environmental Microbiology, September 1998, p. 3389-3396, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5' Nuclease (TaqMan) Assaydagger

R. D. Oberst,1,* M. P. Hays,1 L. K. Bohra,2 R. K. Phebus,2 C. T. Yamashiro,3 C. Paszko-Kolva,3 S. J. A. Flood,3 J. M. Sargeant,1 and J. R. Gillespie1

Food Animal Health & Management Center, College of Veterinary Medicine,1 and Department of Animal Sciences & Industry, College of Agriculture,2 Kansas State University, Manhattan, Kansas 66506, and Perkin-Elmer Corporation, Applied Biosystems Division, Foster City, California 944043

Received 9 February 1998/Accepted 30 June 1998

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC) eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5' nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5' nuclease assay system was sufficient to correctly identify all E. coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed, eaeA-targeted 5' nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 when >= 103 CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when >= 104 CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to >= 102 CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when >= 104 CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5' nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.

* Corresponding author. Mailing address: Food Animal Health & Management Center, College of Veterinary Medicine, Kansas State University, 1800 Denison Ave., VCS Bldg., Manhattan, KS 66506. Phone: (785) 532-4411. Fax: (785) 532-4288. E-mail: oberst{at}vet.ksu.edu.

dagger Contribution no. 98-291-J from the Kansas Agricultural Experiment Station.

Applied and Environmental Microbiology, September 1998, p. 3389-3396, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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