title: Use of clustered regularly interspaced short palindromic repeat sequence polymorphisms for specific detection of enterohemorrhagic Escherichia coli strains of serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by real-time PCR.
abstract: We explored the genetic diversity of the clustered regularly interspaced short palindromic repeat (CRISPR) regions of enterohemorrhagic Escherichia coli (EHEC) to design simplex real-time PCR assays for each of the seven most important EHEC serotypes worldwide. A panel of 958 E. coli strains investigated for their CRISPR loci by high-throughput real-time PCR showed that CRISPR polymorphisms in E. coli strongly correlated with both O:H serotypes and the presence of EHEC virulence factors (stx and eae genes). The CRISPR sequences chosen for simplex real-time PCR amplification of EHEC strains belonging to the top 7 EHEC serogroups differentiated clearly between EHEC and non-EHEC strains. Specificity estimates for the CRISPR PCR assays varied from 97.5% to 100%. Sensitivity estimates for the assays ranged from 95.7% to 100%. The assays targeting EHEC O145:H28, O103:H2, and O45:H2 displayed 100% sensitivity. The combined usage of two simplex PCR assays targeting different sequences of the O26 CRISPR locus allowed detection of EHEC O26:H11 with 100% sensitivity. By combining two simplex PCR assays targeting different sequences of the EHEC O157 CRISPR locus, EHEC O157:H7 was detected with 99.56% sensitivity. EHEC O111:H8 and EHEC O121:H19 were detected with 95.9% and 95.7% sensitivity, respectively. This study demonstrates that the identification of EHEC serotype-specific CRISPR sequences is more specific than the mere identification of O-antigen gene sequences, as is used in current PCR protocols for detection of EHEC strains.
authors.affiliation: Anses (French Agency for Food, Environmental and Occupational Health and Safety), Food Safety Laboratory, Maisons-Alfort, France.
authors.forename: Sabine
authors.initials: S
authors.lastname: Delannoy
authors.forename: Lothar
authors.initials: L
authors.lastname: Beutin
authors.forename: Patrick
authors.initials: P
authors.lastname: Fach
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D054324:Enterohemorrhagic Escherichia coli / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000382:microbiology*; D014644:Genetic Variation; D006801:Humans; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial