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EVALUATION OF RAPID DNA EXTRACTION PROCEDURES FOR THE QUANTITATIVE DETECTION OF FUNGAL CELLS USING REAL TIME PCR ANALYSIS
Citation:
Haugland, R A., N Brinkman, AND S J. Vesper. EVALUATION OF RAPID DNA EXTRACTION PROCEDURES FOR THE QUANTITATIVE DETECTION OF FUNGAL CELLS USING REAL TIME PCR ANALYSIS. JOURNAL OF MICROBIOLOGICAL METHODS 50(3):319-323, (2002).
Impact/Purpose:
1. Evaluate the efficacy of QPCR technology for detecting low level microbiological contaminants in water supplies
2. Provide additional data on the range of pathogenic or potentially pathogenic species of fungi in community distribution system and hospital water samples.
Description:
The ease and rapidity of quantitative DNA sequence detection by real-time PCR instruments promises to make their use increasingly common for the microbial analysis many different types of environmental samples. To fully exploit the capabilities of these instruments, correspondingly rapid, easy and reproducible methods for extracting microbial DNA from these samples need to be identified or developed. To address this need, we evaluated three new methods for the extraction of DNA from fungal cells that offer significant savings in time and effort over our previously reported procedures. Existing or newly developed TaqMan PCR assays for four fungal species: Aspergillus fumigatus, Stachybotrys chartarum, Candida albicans and Geotrichum candidum; and an established comparative threshold quantitative analysis method were used to determine the extraction methods' yields of target DNA from known cells in the presence of different, commercially available lysis reagents gave similar DNA yields to those of our most efficient previous method while the third, which did not use bead milling gave significantly lower yields. Both of the bead milling methods were found to support accurate and precise quantification of target fugal cells in air and water samples, however only one of these methods, involving further purification of the DNA by a streamlined silica adsorption procedure was found to be adequate for the analysis of building dust samples.