title: Development of a rapid microarray-based DNA subtyping assay for the alleles of Shiga toxins 1 and 2 of Escherichia coli.
abstract: In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli (STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise.
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, Germany lutz.geue@fli.bund.de.
authors.forename: Lutz
authors.initials: L
authors.lastname: Geue
authors.affiliation: Alere Technologies GmbH, Jena, Germany Technische Universität Dresden, Institute for Medical Microbiology, Virology and Hygiene, Dresden, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Stieber
authors.affiliation: Alere Technologies GmbH, Jena, Germany Technische Universität Dresden, Institute for Medical Microbiology, Virology and Hygiene, Dresden, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Monecke
authors.affiliation: Alere Technologies GmbH, Jena, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Engelmann
authors.affiliation: Technische Universität Dresden, Institute for Medical Microbiology, Virology and Hygiene, Dresden, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Gunzer
authors.affiliation: Alere Technologies GmbH, Jena, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Slickers
authors.affiliation: Alere Technologies GmbH, Jena, Germany.
authors.forename: Sascha D
authors.initials: SD
authors.lastname: Braun
authors.affiliation: Alere Technologies GmbH, Jena, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Ehricht
mesh_terms: D000818:Animals; D057205:Automation, Laboratory / Q000379:methods; D019295:Computational Biology / Q000379:methods; D004927:Escherichia coli Infections / Q000382:microbiology / Q000662:veterinary; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D022622:Shiga Toxin 1 / Q000235:genetics*; D022641:Shiga Toxin 2 / Q000235:genetics*; D054323:Shiga-Toxigenic Escherichia coli / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D012984:Software
chemical_list: D022622:Shiga Toxin 1; D022641:Shiga Toxin 2