PCR-based DNA amplification and presumptive detection of Escherichia coli O157:H7 with an internal fluorogenic probe and the 5' nuclease (TaqMan) assay

R. D. Oberst, M. P. Hays, L. K. Bohra, R. K. Phebus, C. T. Yamashiro, C. Paszko-Kolva, S. J.A. Flood, J. M. Sargeant, J. R. Gillespie

Research output: Contribution to journalArticle

154 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3389-3396
Number of pages8
JournalApplied and environmental microbiology
Volume64
Issue number9
DOIs
StatePublished - Jan 1 1998

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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    Oberst, R. D., Hays, M. P., Bohra, L. K., Phebus, R. K., Yamashiro, C. T., Paszko-Kolva, C., Flood, S. J. A., Sargeant, J. M., & Gillespie, J. R. (1998). PCR-based DNA amplification and presumptive detection of Escherichia coli O157:H7 with an internal fluorogenic probe and the 5' nuclease (TaqMan) assay. Applied and environmental microbiology, 64(9), 3389-3396. https://doi.org/10.1128/aem.64.9.3389-3396.1998