hybridization of whole cells with rRNA-targeted, fluorescently labelled oligonucleotide probes is a powerful method to specifically detect micro-organisms in their natural habitat without cultivation and subsequent identification by phenotypic characterization. To examine the use of this method for the specific detection of pathogenic species, we have designed an oligonucleotide probe which binds to the 18S rRNA of and the two most important pathogenic species, and differentiates them from other clinically relevant species. After establishing suitable hybridization conditions, we confirmed the specificity of our probe O20 in RNA dot blot hybridizations with a series of reference strains and clinical isolates of medically important species. All and strains hybridized with the probe, whereas all strains of and did not. When we used the fluorescently labelled probe O20 to specifically detect single cells of the two target species by hybridization, both and reacted strongly with the probe and could be clearly differentiated from and although the latter organism contains only two nucleotide mismatches in the probe target region. This discrimination capacity was also seen when mixed suspensions of and were hybridized with the probe. After infection of a human endothelial cell line with and cells adhering to the endothelial cells were easily distinguishable from the cells by fluorescent hybridization with probe O20. In addition, germ tubes and hyphae of were also efficiently labelled. The application of fluorescently labelled rRNA-targeted oligonucleotide probes therefore appears to be a valuable tool for the specific detection and identification of different members of the genus which does not require any cultivation.


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