| Summary: | 博士 === 國立成功大學 === 醫學工程研究所碩博士班 === 95 === Infections caused by yeasts have increased in recent decades due to the increasing population of immunocompromised patients. In addition to Candida spp., infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces have been widely reported. Accurate and rapid identification of yeast pathogens is important for appropriate treatment of patients with antifungal agents. The objectives of this study were to evaluate the feasibility of using the sequence of ribosomal DNA internal transcribed spacer (ITS) regions and an oligonucleotide array to identify clinically important yeasts. The first part of this thesis evaluated the feasibility of ITS sequencing for yeast identification. Both ITS1 and ITS2 regions of 373 strains (86 species) including 299 reference strains and 74 clinical isolates were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database using BLAST (Basic Local Alignment Search Tool) for species determination. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. The second part of this thesis was to evaluate primer labeling (one or both primers) and the use of sense or antisense probe on the hybridization signal. It was found that labeling of both primers produced, under most conditions, stronger hybridization signal. However, the effect of using sense or antisense probe on hybridization signal was not predictable. The third part of this thesis was to develop an oligonucleotide array to identify 77 species (16 genera) of clinically relevant yeasts based on the ITS sequence. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In addition, 42 yeast-positive blood cultures were analyzed by the oligonucleotide array, and all specimens were correctly identified. In conclusion, yeast identification by both methods (sequence analysis and oligonucleotide array) is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h starting from isolated colonies.
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