BeschreibungAccuracy coupled with speed is of greatest importance in diagnostics of infectious disease. This applies in particular e.g. when it comes to identifying antibiotic resistance of infection causing microbes in patients already under bad health conditions (immunocompromised, intensive care unit). In contrast to conventional diagnostic methods lasting at least 24 hrs due to their requirement for microbial growth, DNA- and PCR (polymerase chain reaction) -based methods meet here the needs for a fast, reliable and thereby life-saving diagnosis. We present here a novel approach for fast identification of antibiotic resistance by combining PCR amplification with subsequent microarray analysis offering the advantage of parallel target testing compared to conventional PCR diagnosis. As proof of concept we established a prototype microarray for the detection of 12 different antibiotic resistance genes (ABR-ARChipTM). The diagnostic test is based on a multiplex-PCR from genomic bacterial DNA, the incorporation of fluorescently labelled nucleotides (dCTP-Cy5) during a primer extension step, the subsequent hybridization onto a glass slide microarray containing specific oligonucleotide probes for the resistance genes of interest and the final detection of labelled, microarray-bound target by a 635nm/ 532nm dual-wavelength laser scanner. The ABR-ARChipTM was tested with genomic DNA from numerous clinical isolates of Staphylococcus epidermis showing various spectra of antibiotic resistance. Results obtained by microarray analysis were compared with those from conventional, phenotypic antibiotic resistance analysis performed by disk diffusion testing. Our studies show the potential and usefulness of DNA microarrays for genotypic antibiotic resistance testing. In parallel to the ABR-ARChipTM prototype currently adapted to specific clinical settings, we have recently started to develop a pathogen detection microarray including -at this initial stage- oligonucleotide probes for Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecium and Enterococcus faecalis - representing some of the most frequent pathogens in community- and hospital-acquired infections. From our preliminary results we conclude that DNA microarrays are useful and promising tools for rapid antibiotic resistance determination as well as pathogen identification. When applied in combination they are certainly most effective in infectious disease management.
|3 Feb. 2003
|V. Annual Linz Winter Workshop ( Single Molecule Techniques in Biophysics and Drug Discovery)
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