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

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A Correction to this article was published on 24 August 2019

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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.

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Change history

  • 24 August 2019

    The original version of this article contained a mistake in the co-author���s conflict of interest statement. Erika von Mutius wishes to add the following disclosures: ���E. von Mutius is listed as an inventor on the following patents: publication number EP 1411977, composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases, granted on 18 April 2007; publication number EP1637147, stable dust extract for allergy protection, granted on 10 December 2008; publication number EP 1964570, pharmaceutical compound to protect against allergies and inflammatory diseases, granted on 21 November 2012. E. von Mutius is listed as inventor and has received royalties on the following patent: publication number EP2361632, specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders, granted on 19 March 2014���.

  • 24 August 2019

    The original version of this article contained a mistake in the co-author���s conflict of interest statement. Erika von Mutius wishes to add the following disclosures: ���E. von Mutius is listed as an inventor on the following patents: publication number EP 1411977, composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases, granted on 18 April 2007; publication number EP1637147, stable dust extract for allergy protection, granted on 10 December 2008; publication number EP 1964570, pharmaceutical compound to protect against allergies and inflammatory diseases, granted on 21 November 2012. E. von Mutius is listed as inventor and has received royalties on the following patent: publication number EP2361632, specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders, granted on 19 March 2014���.

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

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Acknowledgments

This study was supported by “Sonderforschungsbereich Transregio 22” from the Deutsche Forschungsgesellschaft. We gratefully thank Ms. Barbara Fritz from the Technische Universität München (TUM) for excellent technical assistance as well as the TUM Graduate School for backup support.

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Authors have no conflict of interest

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Financial support by the Deutsche Forschungsgesellschaft (SFB/TR 22).

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Authors and Affiliations

  1. Institute of Animal Hygiene, Technische Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany

    Tobias Janke, Karin Schwaiger, Carmen Fahn, Johann Bauer & Melanie Mayer

  2. University Children’s Hospital Munich, Lindwurmstraße 4, 80337, Munich, Germany

    Markus Ege & Erika von Mutius

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  1. Tobias Janke
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Correspondence to Tobias Janke.

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Janke, T., Schwaiger, K., Ege, M. et al. Analysis of the Fungal Flora in Environmental Dust Samples by PCR–SSCP Method. Curr Microbiol 67, 156–169 (2013). https://doi.org/10.1007/s00284-013-0344-3

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