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High-Throughput Detection of Pathogenic Yeasts of the Genus Trichosporon

Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, Florida 33149

Received 4 February 2004/ Returned for modification 4 April 2004/ Accepted 6 May 2004

The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon. This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 10² genome molecules in PCRs and 10⁷ to 10⁸ molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.

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