High-Throughput Detection of Pathogenic Yeasts of the Genus Trichosporon

Abstract

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.

Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients

“…33,34 To assess this notion, clinical implementation of methods facilitating reliable identification of these species at an adequate level of sensitivity would be required. A number of methods for rapid typing of fungal pathogens have been published, [35][36][37][38][39][40][41][42][43][44][45] and the development of novel and expectedly more potent methods is currently underway, including also approaches based on the exploitation of nano-technological devices, as currently pursued in our laboratory. Rapid and sensitive techniques for the identification of putatively non-or low-pathogenic environmental fungal species that may cause clinically silent fungemia could improve the interpretation of results obtained by broad-spectrum PCR screening methods.…”

Temporal and spatial distributions of marine Synechococcus in the Southern California Bight assessed by hybridization to bead-arrays

“…3c,d). As PCR efficiency was similar for control reactions, the differences in hybridization signal were likely due to the capacity of a given probe to hybridize efficiently to its target, which could be affected by secondary structure of the target or probe, or the density of the probes coupled onto the beads (Diaz & Fell 2004). The average recorded PE signal (on an arbitrary scale) for probe IV-A ranged from 50 (for at least a 2-fold signal above background) to 1250, with a signal:background ratio of 50.…”

Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand

“…Probes had Tm's higher than 55°C, GC contents around 50%, minimum secondary structure and primer dimer potential, and lengths between 20-25 base pairs. Basepair differences conferring specificity were located near the center of the probe (Diaz and Fell, 2004). Probes were manufactured with a C-12 modifier on the 5′ end (Integrated DNA Technologies) to reduce steric hindrance from microspheres.…”

“…Probes were covalently coupled to 5.6μm, polystyrene, carboxylated beads (Miraibio, Alameda, CA) by a carbodiimide coupling method (Diaz and Fell, 2004; http://www.miraibio.com) with slight modifications. The uncoupled bead stock was vortexed and sonicated for 20 s and 200μl (5 × 10 6 beads) was transferred to a low binding 1.5 ml microcentrifuge tube (USA Scientific, Ocala, FL).…”