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

J Med Microbiol

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

Published: September 2006

AI Article Synopsis

  • Yeasts from the Malassezia genus are linked to various skin disorders in humans and animals, prompting a need for advanced identification methods due to the discovery of new species.
  • A new bead suspension culture-based array combines nucleic acid hybridization analysis with Luminex technology for rapid and accurate species identification, using specific fluorescent probes based on rRNA sequences.
  • This high-throughput array allows for multiplex detection, can differentiate between target and non-target sequences with precision, and can be expanded to include more pathogenic species, streamlining the diagnostic process.

Article Abstract

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.

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Source
http://dx.doi.org/10.1099/jmm.0.46630-0DOI Listing

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