PCR-Based DNA Amplification and Presumptive Detection ofEscherichia coliO157:H7 with an Internal Fluorogenic Probe and the 5′ Nuclease (TaqMan) Assay

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Abstract
Presumptive identification ofEscherichia coliO157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagicE. coli(EHEC)eaeAgene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detectingE. coliO157:H7 DNA. The specificity of theeaeA-based 5′ nuclease assay system was sufficient to correctly identify allE. coliO157: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 ofE. coliO157:H7 when ≥103CFU/ml was present in modified tryptic soy broth (mTSB) or modifiedE. colibroth and when ≥104CFU/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 ≥102CFU/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 ≥104CFU/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 ofE. coliO157:H7 in ground beef and potentially in other food and environmental samples.