Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis.
Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartarum, Cladosporium herbarum and Alternaria alternata. The extraction methods differed in their use of different cell lysis procedures. These included grinding in liquid nitrogen, grinding at ambient temperature, sonication, glass bead milling and freeze-thawing. DNA purification and recovery from the lysates were performed using a commercially available system based on the selective binding of nucleic acids to glass milk. A simple quantitative competitive polymerase chain reaction (QC-PCR) assay was developed for use in determining copy numbers of the internal transcribed spacer (ITS) regions of the ribosomal RNA operon (rDNA) in the total DNA extracts. These quantitative analyses demonstrated that the method using glass bead milling was most effective in recovering PCR templates from each of the different types of conidia both in terms of absolute copy numbers recovered and also in terms of lowest extract to extract variability. Calculations of average template copy yield per conidium in this study indicate that the bead milling method is sufficient to support the detection of less than ten conidia of each of the different organisms in a PCR assay.
Haugland
R A
RA
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268, USA. haugland.rich@epa.gov
Heckman
J L
JL
Wymer
L J
LJ
D004271:DNA, Fungal / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000302:isolation & purification; D003904:Mitosporic Fungi / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004271:DNA, Fungal; D004275:DNA, Ribosomal