title: Rapid Assays for Specific Detection of Fungi of Scopulariopsis and Microascus Genera and Scopulariopsis brevicaulis Species.
abstract: OBJECTIVE
  Fungi of Scopulariopsis and Microascus genera cause a wide range of infections, with S. brevicaulis being the most prevalent aetiological agent of mould onychomycosis. Proper identification of these pathogens requires sporulating culture, which considerably delays the diagnosis. So far, sequencing of rDNA regions of clinical isolates has produced ambiguous results due to the lack of reference sequences in publicly available databases. Thus, there is a clear need for the development of new molecular methods that would provide simple, rapid and highly specific identification of Scopulariopsis and Microascus species. The objective of this study was to develop simple and fast assays based on PCR and real-time PCR for specific detection of fungi from Scopulariopsis and Microascus genera, and separately, S. brevicaulis species.
  
  
  METHODS
  On the basis of alignment of β-tubulin gene sequences, Microascus/Scopulariopsis-specific primers were designed and S. brevicaulis-specific primers were reevaluated. DNA from cultured fungal isolates, extracted in a two-step procedure, was used in Microascus/Scopulariopsis-specific and S. brevicaulis-specific PCR and real-time PCR followed by electrophoresis or melting temperature analysis, respectively.
  
  
  RESULTS
  The specificity of the assays was confirmed, as positive results were obtained only for Scopulariopsis spp. and Microascus spp. isolates tested in Microascus/Scopulariopsis-specific assay, and only for S. brevicaulis and S. koningii (syn. S. brevicaulis) isolates in a S. brevicaulis-specific assay, respectively, and no positive results were obtained neither for other moulds, dermatophytes, yeast-like fungi, nor for human DNA.
  
  
  CONCLUSIONS
  The developed assays enable fast and unambiguous identification of Microascus spp. and Scopulariopsis spp. pathogens.
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdańsk University of Technology, Narutowicza 11/12, 80-233, Gdańsk, Poland. milena.kordalewska@pg.gda.pl.
authors.forename: Milena
authors.initials: M
authors.lastname: Kordalewska
authors.affiliation: Department of Applied Microbiology, Faculty of Biology, Institute of Microbiology, University of Warsaw, Miecznikowa 1, 02-096, Warsaw, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Jagielski
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdańsk University of Technology, Narutowicza 11/12, 80-233, Gdańsk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Brillowska-Dąbrowska
mesh_terms: D001203:Ascomycota / Q000145:classification* / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D003881:Dermatomycoses / Q000175:diagnosis* / Q000382:microbiology*; D004586:Electrophoresis; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D044366:Transition Temperature; D014404:Tubulin / Q000235:genetics
chemical_list: D017931:DNA Primers; D014404:Tubulin
keywords: Detection; Identification; Microascus; PCR; Real-time PCR; Scopulariopsis