High-throughput detection of pathogenic yeasts of the genus trichosporon.
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 10(2) genome molecules in PCRs and 10(7) to 10(8) 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.
Diaz
Mara R
MR
Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA. mdiaz@rsmas.miami.edu
Fell
Jack W
JW
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004271:DNA, Fungal / Q000302:isolation & purification; D006801:Humans; D009181:Mycoses / Q000175:diagnosis*; D009693:Nucleic Acid Hybridization; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D014250:Trichosporon / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity*
D017931:DNA Primers; D004271:DNA, Fungal