Skip directly to search Skip directly to A to Z list Skip directly to page options Skip directly to site content

NIOSHTIC-2 Publications Search

Search Results

Comparison of DNA extraction methodologies used for assessing fungal diversity via ITS sequencing.

Authors
Rittenour-WR; Park-J-H; Cox-Ganser-JM; Beezhold-DH; Green-BJ
Source
J Environ Monit 2012 Mar; 14(3):766-774
NIOSHTIC No.
20040217
Abstract
Traditional methods of assessing fungal exposure have been confounded by a number of limiting variables. The recent utilization of molecular methods such as internal transcribed spacer (ITS) sequencing of ribosomal RNA genes has provided improved insight into the diversity of fungal bioaerosols in indoor, outdoor and occupational environments. However, ITS analyses may also be confounded by a number of methodological limitations. In this study, we have optimized this technology for use in occupational or environmental studies. Three commonly used DNA extraction methodologies (UltraClean Soil kit, High Pure PCR Template kit, and EluQuik/DNeasy kit) were compared in terms of sensitivity and susceptibility to PCR inhibitors in dust for three common fungal bioaerosols, Aspergillus versicolor, Rhizopus microsporus and Wallemia sebi. Environmental dust samples were then studied using each extraction methodology and results were compared to viable culture data. The extraction methods differed in terms of their ability to efficiently extract DNA from particular species of fungi (e.g. Aspergillus versicolor). In addition, the ability to remove PCR inhibitors from dust samples was most effective using the soil DNA extraction kit. The species composition varied greatly between ITS clone libraries generated with the different DNA extraction kits. However, compared to viable culture data, ITS clone libraries included additional fungal species that are incapable of growth on solid culture medium. Collectively, our data indicated that DNA extraction methodologies used in ITS sequencing studies of occupational or environmental dust samples can greatly influence the fungal species that are detected.
Keywords
Fungi; Exposure-levels; Models; Molecular-structure; Molecular-biology; Genes; Biological-factors; Indoor-air-pollution; Air-quality; Outdoors; Environmental-exposure; Environmental-factors; Dusts; Dust-exposure; Dust-analysis; Indoor-environmental-quality
Contact
William R. Rittenour, Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Rd., Morgantown, West Virginia, 26505
CODEN
JEMOFW
Publication Date
20120301
Document Type
Journal Article
Email Address
ikx8@cdc
Fiscal Year
2012
NTIS Accession No.
NTIS Price
Identifying No.
B02012012
Issue of Publication
2
ISSN
1464-0325
NIOSH Division
HELD; DRDS
Priority Area
Healthcare and Social Assistance; Services
Source Name
Journal of Environmental Monitoring
State
WV; TN
TOP