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J Med Microbiol 55 (2006), 1197-1209; DOI: 10.1099/jmm.0.46630-0
© 2006 Society for General Microbiology
ISSN 1473-5644

Microcoding and flow cytometry as a high-throughput fungal identification system for Malassezia species

Mara R. Diaz1, Teun Boekhout2, Bart Theelen2, Marjan Bovers2, Francisco J. Cabañes3 and Jack W. Fell1

1 Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA

2 Centraalbureau voor Schimmelcultures, Yeast Division, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

3 Grup de Micologia Veterinària, Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Bellaterra, Barcelona E-08193, Spain

Correspondence
Mara R. Diaz
mdiaz{at}rsmas.miami.edu

Received 15 March 2006
Accepted 22 May 2006

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.

Abbreviations: AFLP, amplified fragment length polymorphism; ITS, internal transcribed spacer; MFI, median fluorescence intensity.




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