Current Microbiology

, Volume 67, Issue 2, pp 156–169

Analysis of the Fungal Flora in Environmental Dust Samples by PCR–SSCP Method

  • Tobias Janke
  • Karin Schwaiger
  • Markus Ege
  • Carmen Fahn
  • Erika von Mutius
  • Johann Bauer
  • Melanie Mayer
Article

DOI: 10.1007/s00284-013-0344-3

Cite this article as:
Janke, T., Schwaiger, K., Ege, M. et al. Curr Microbiol (2013) 67: 156. doi:10.1007/s00284-013-0344-3

Abstract

Conventional microbiological techniques yield only limited information on the composition of fungal communities in dust. The aim of this study was to establish and optimize PCR-single strand conformation polymorphism (PCR–SSCP) analysis for investigation of fungal diversity in rural dust samples. Three different DNA extraction protocols were tested on 38 fungal cultures. A total of six known universal fungal primer pairs were tested targeting the 18S rRNA gene, the 28S rRNA gene and the ITS region, respectively. Objective evaluation was performed with respect to the following parameters: efficiency to amplify all 38 strains; separation of seven species from different phylogenetic groups on the SSCP gel; additional bands in PCR–SSCP analysis; possibility to classify the amplified gene fragments to species level. Primer ITS1/ITS4 and PowerSoil™ DNA isolation showed the best performance in most cases and were chosen for further analysis. The detection limit of the developed system was 200 CFU/g dust. Moreover, the reproducibility of the system could be demonstrated, leading to average profile similarities of 94.94 % [SD = 2.51] within gels, 93.03 % [SD = 4.69] between different days and 87.66 % [SD = 6.62] between different gels when testing shed and mattress dust samples. Sequencing allowed identification on species level, in detail: Alternaria alternata, Cladosporium sphaerospermum, Cladosporium cladosporioides as well as the yeasts Candida cabralensis and Candida catenulata. This demonstrates the adaptability of the method. In this study, a standardized system for fungal community analysis was developed that provides reproducible results applicable for epidemiological purposes.

Abbreviations

ARISA

Automated ribosomal intergenic spacer analysis

CFU

Colony forming unit

CTAB

Cetyltrimethylammoniumbromid

DGGE

Denaturing gradient gel electrophoresis

DSMZ

German collection of microorganisms and cell cultures

EPS

Exopolysaccharide

ITS

Internal transcribed spacer

LSU

Large-subunit

PCR

Polymerase chain reaction

SSCP

Single-strand conformation polymorphism

SSU

Small-subunit

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Tobias Janke
    • 1
  • Karin Schwaiger
    • 1
  • Markus Ege
    • 2
  • Carmen Fahn
    • 1
  • Erika von Mutius
    • 2
  • Johann Bauer
    • 1
  • Melanie Mayer
    • 1
  1. 1.Institute of Animal HygieneTechnische Universität MünchenFreisingGermany
  2. 2.University Children’s Hospital MunichMunichGermany