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Development of a fungus-specific PCR assay for detecting low-level fungi in an indoor environment.

Authors
Zhou-G; Whong-WZ; Ong-T; Chen-B
Source
Mol Cell Probes 2000 Dec; 14(6):339-348
NIOSHTIC No.
20020872
Abstract
A fungus-specific PCR assay using only one primer set has been developed for detecting indoor fungi. Four fungal primer sets, NS3/NS4, NS5/NS6, FF1/FR1 and FF2/FR1, were tested with DNA from humans, rats, mice, bacteria, pollens and six commonly found fungal species (Alternaria chamydospora, Aspergillus flavus, Candida famata, Cladosporium fermentans, Penicillium chrycoIgenum and Stachybotrys chartarum). Results indicated that, although all four primer sets could amplify the fungal DNA, only FF2/FR1 demonstrated no cross-amplification with non-fungal DNA. In addition, these amplified fragments were sequenced to ensure that they indeed matched known fungal DNA sequences. Furthermore, besides the tested fungi, eighteen more genera of fungal sequences were examined and found to match the FF2/FR1. Here, the method of bead-beating was identified as the most effective way for spore breakage and fungal DNA release. The PCR amplification efficiency and potential inhibition were examined using different process solutions and preparation procedures. It was found that, when using 20% nutrient media and homogenization-first procedure, a higher amplification efficiency with less inhibition was achieved. Although positive bands were observed at 0.2 fungal spore/reaction using the homogenization-first procedure, the sensitivity of this assay would be two fungal spores/reaction for environmental samples.
Keywords
Fungicides; Fungi; Environmental-factors; Laboratory-animals; Animals; Animal-studies; Bacterial-cultures; Bacteria; Sampling; Microorganisms
Contact
Health Effects Laboratory Division, NIOSH, 1095 Willowdale Road, Morgantown, WV 26505, USA
Publication Date
20001201
Document Type
Journal Article
Email Address
bdc4@cdc.gov
Fiscal Year
2001
NTIS Accession No.
NTIS Price
Issue of Publication
6
ISSN
0890-8508
NIOSH Division
HELD
Source Name
Molecular and Cellular Probes
State
WV
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