title: A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens.
abstract: A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.
authors.affiliation: Department of Bioresources, ARC Seibersdorf Research GmbH, A-2444 Seibersdorf, Austria.
authors.forename: Tanja
authors.initials: T
authors.lastname: Kostić
authors.forename: Alexandra
authors.initials: A
authors.lastname: Weilharter
authors.forename: Salvatore
authors.initials: S
authors.lastname: Rubino
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Delogu
authors.forename: Sergio
authors.initials: S
authors.lastname: Uzzau
authors.forename: Knut
authors.initials: K
authors.lastname: Rudi
authors.forename: Angela
authors.initials: A
authors.lastname: Sessitsch
authors.forename: Levente
authors.initials: L
authors.lastname: Bodrossy
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D015373:Bacterial Typing Techniques / Q000379:methods; D001483:Base Sequence; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D027081:DNA Gyrase