A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of non-pathogens

A major challenge in microbial diagnostics is the parallel detection and identiWcation of low-bundance pathogens within a complex
microbial community. In addition, a high speciWcity providing robust, reliable identiWcation 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 speciWcity of the assay. Our approach enabled the
sensitive and speciWc 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, signiWcantly 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