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A RAPID METHOD FOR THE EXTRACTION OF FUNGAL DNA FROM ENVIRONMENTAL SAMPLES: EVALUATION IN THE QUANTITATIVE ANALYSIS OF MEMNONIELLA ECHINATA CONIDIA USING REAL TIME DETECTION OF PCR PRODUCTS
Citation:
Haugland, R A., S J. Vesper, AND L J. Wymer. A RAPID METHOD FOR THE EXTRACTION OF FUNGAL DNA FROM ENVIRONMENTAL SAMPLES: EVALUATION IN THE QUANTITATIVE ANALYSIS OF MEMNONIELLA ECHINATA CONIDIA USING REAL TIME DETECTION OF PCR PRODUCTS. Presented at Mycologcal Society of American Annual Meeting, Burlington, VT, July 29-August 3, 2000.
Impact/Purpose:
To understand children's risks from exposure to molds in their environment and to explore risk management options for mitigating those risks.
Description:
New technologies are creating the potential for using nucleic acid sequence detection to perform routine microbiological analyses of environmental samples. Our laboratory has recently reported on the development of a method for the quantitative detection of Stachybotrys chartarum conidia in air and indoor dust samples using a primer and probe set that is specific for ITS region rDNA sequences from this organism and the TaqMan fluorigenic probe assay. This system allows analyses of up to 96 samples to be performed in two hours. The high sample throughput permitted by this system has led us to search for more streamlined methods for preparing suitable DNA extracts for analysis. In the current study we describe a modification of our previously reported method for the extraction of DNA from fungal conidia that reduces the overall sample processing time by approximately one-half. The new method continues to use bead milling to disrupt the fungal cells but substitutes a commercially available manifold-based DNA purification system for the previously used centrifugation-based system. We also describe a new TaqMan primer and probe set for the specific detection of the toxigenic fungal species, Memnoniella echinata. DNA extractions by the new method were found to support similar levels of accuracy and precision in the quantification of M. echinata conidia in both liquid and dust matrices to those previously reported for S. chartarum using the longer extraction method. Ninety-five percent occurrence ranges of these quantitative estimates were within 50 to 200% of actual cell numbers in the liquid samples and 20 to 65% in the dust samples over a range from 25 to 1000 cells per sample.