High through-put detection of pathogenic yeasts in the genus trichosporon

摘要

The emergence of opportunistic and antifungal resistant strains has given rise to an urgent need for a rapid and accurate method for the detection of fungal pathogens. In this application, we demonstrate the detection of medically important fungal pathogens at the species level. The present 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 the fluorescent detection with a 532 nm laser. Using this technology we designed and tested various multiplex formats, the performance of forty eight species specific and group specific capture probes designed from sequence analysis in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions (ITS), and intergenic spacer region (IGS). Species-specific biotinylated amplicons (>600 bp) were generated with three sets of primers to yield fragments from the three regions. The developed assay was specific and relatively fast, as it discriminated species differing by one nucleotide and required less than 50 min following amplification to process a 96 well plate with the capability to detect up to 100 species per well. The sensitivity of the assay allowed the detection as low as 10² genome molecules in PCR reactions and 10⁷ to 10⁸ molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species and had the flexibility to identify species in a multiplex format by combining different sets of beads. The assay can be expanded to include all known pathogenic fungal species.