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Curr Microbiol (2013) 67:156169
DOI 10.1007/s00284-013-0344-3
Analysis of the Fungal Flora in Environmental Dust Samples by PCRSSCP Method
Tobias Janke Karin Schwaiger Markus Ege
Carmen Fahn Erika von Mutius Johann Bauer
Melanie Mayer
Received: 2 July 2012 / Accepted: 22 February 2013 / Published online: 10 March 2013 Springer Science+Business Media New York 2013
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 (PCRSSCP) 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: efciency to amplify all 38 strains; separation of seven species from different phylogenetic groups on the SSCP gel; additional bands in PCRSSCP analysis; possibility to classify the amplied gene fragments to species level. Primer ITS1/ ITS4 and PowerSoilTM 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 prole 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 identication 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.
AbbreviationsARISA Automated ribosomal intergenic spacer analysis CFU Colony forming unitCTAB CetyltrimethylammoniumbromidDGGE Denaturing gradient gel electrophoresisDSMZ German collection of microorganisms and cell culturesEPS ExopolysaccharideITS Internal transcribed spacerLSU Large-subunitPCR Polymerase chain reactionSSCP Single-strand conformation polymorphismSSU Small-subunit
Introduction
During recent years, there has been an increasing interest in the environmental exposure to microorganisms in air and
T. Janke (&) K. Schwaiger C. Fahn J. Bauer M. Mayer
Institute of Animal Hygiene, Technische Universitat Mnchen, Weihenstephaner Berg...