Enterohemorrhagic Escherichia coli O157:H7 is responsible for many outbreaks of gastrointestinal illness and hemolytic uremic syndrome worldwide. Monitoring this pathogen in food and water supplies is an important public health issue. Highly conserved genetic markers, which are characteristic for specific strains, can provide direct identification of target pathogens. In this study, we examined a new detection strategy for pathogenic strains of E. coli O157:H7 serotype based on a conserved signature insertion/deletion (CSI) located in the ybiX gene using TaqMan-probe-based quantitative PCR (qPCR). The qPCR assay was linear from 1.0 × 10(2) to 1.0 × 10(7) genome copies and was specific to O157:H7 when tested against a panel of 15 non-O157:H7 E. coli. The assay also maintained detection sensitivity in the presence of competing E. coli K-12, heterologous nontarget DNA spiked in at a 1000-fold and 800-fold excess of target DNA, respectively, demonstrating the assay's ability to detect E. coli O157:H7 in the presence of high levels of background DNA. This study thus validates the use of strain-specific CSIs as a new class of diagnostic marker for pathogen detection.