title: Insertion/deletion-based approach for the detection of Escherichia coli O157:H7 in freshwater environments.
abstract: 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.
authors.affiliation: Department of Biology, McMaster University , Life Sciences Building, 1280 Main St. West, Hamilton, Ontario, Canada L8S 4K1.
authors.forename: Shirley Y
authors.initials: SY
authors.lastname: Wong
authors.forename: Athanasios
authors.initials: A
authors.lastname: Paschos
authors.forename: Radhey S
authors.initials: RS
authors.lastname: Gupta
authors.forename: Herb E
authors.initials: HE
authors.lastname: Schellhorn
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay; D017931:DNA Primers / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005618:Fresh Water / Q000382:microbiology*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins