PCR-BASED DNA AMPLIFICATION AND PRESUMPTIVE DETECTION OF ESCHERICHIA COLI0157-H7 WITH AN INTERNAL FLUOROGENIC PROBE AND THE 5'-NUCLEASE (TAQMAN) ASSAY

Presumptive identification of Escherichia coil O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohe morrhagic E. coli (EHEC) eaeA gene, In this report, we describe the de velopment and evaluation of the sensitivity and specificity of a PCR-b ased 5' nuclease assay for presumptively detecting E. coli O157:H7 DNA . The specificity of the eaeA-based 5' nuclease assay system was suffi cient to correctly identify all E. coli O157:H7 strains evaluated, mir roring the previously described specificity of the PCR primers. The SZ -primed, eaeA-targeted 5' nuclease detection assay was capable of rapi d, semiautomated, presumptive detection of E. coli O157:H7 when greate r than or equal to 10(3) CFU/ml was present in modified tryptic soy br oth (mTSB) or modified E. coli broth and when greater than or equal to 10(4) CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enric hment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen ), improved the detection threshold to greater than or equal to 10(2) CFU/ml. Surprisingly, immediately after IMS, the sensitivity of cultur ing on sorbitol MacConkey agar containing cefeximine and tellurite (CT -SMAC) was such that identifiable colonies were demonstrated only when greater than or equal to 10(4) CFU/ml was present in the sample. Seve ral factors that might be involved in creating these false-negative CT -SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5' n uclease detection system demonstrated that it can be integrated readil y 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 acid envi ronmental samples.