Microcoding and flow cytometry as a high-throughput fungal identification system for Malassezia species.
Yeasts of the genus Malassezia have been associated with a variety of dermatological disorders in humans and domestic animals. With the recent recognition of new members of the genus, new questions are emerging with regard to the pathogenesis and epidemiology of the new species. As new species are recognized, a precise and comprehensive identification system is needed. Herein is described a bead suspension culture-based array that combines the specificity and reliability of nucleic acid hybridization analysis with the speed and sensitivity of the Luminex analyser. The developed 16-plex array consisted of species- and group-specific capture probes that acted as 'microcodes' for species identification. The probes, which were designed from sequence analysis in the D1/D2 region of rRNA and internal transcribed spacer (ITS) regions, were covalently bound to unique sets of fluorescent beads. Upon hybridization, the biotinylated amplicon was detected by the addition of a fluorochrome coupled to a reporter molecule. The hybridized beads were subsequently analysed by flow cytometric techniques. The developed array, which allowed the detection of species in a multiplex and high-throughput format, was accurate and fast, since it allowed precise identification of species and required less than 1 h following PCR amplification. The described protocol, which can integrate uniplex or multiplex PCR reactions, permitted the simultaneous detection of target sequences in a single reaction, and allowed single mismatch discrimination between probe and non-target sequences. The assay has the capability to be expanded to include other medically important pathogenic species in a single or multiplex array format.
Diaz
Mara R
MR
Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA.
Boekhout
Teun
T
Centraalbureau voor Schimmelcultures, Yeast Division, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Theelen
Bart
B
Centraalbureau voor Schimmelcultures, Yeast Division, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Bovers
Marjan
M
Centraalbureau voor Schimmelcultures, Yeast Division, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Cabañes
Francisco J
FJ
Grup de Micologia Veterinària, Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Bellaterra, Barcelona E-08193, Spain.
Fell
Jack W
JW
Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA.
D000818:Animals; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D003881:Dermatomycoses / Q000382:microbiology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008290:Malassezia / Q000145:classification* / Q000235:genetics; D008297:Male; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer; D015345:Oligonucleotide Probes