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 effectivenes s in recovering PCR templates from the conidia of a series of fungal specie s often encountered in indoor air. The test organisms were Aspergillus vers icolor, Penicillium chrysogenum, Stachybotrys chartarum, Cladosporium herba rum and Alternaria alternata. The extraction methods differed in their use of different cell lysis procedures. These included grinding in liquid nitro gen, grinding at ambient temperature, sonication, glass bead milling and fr eeze-thawing. DNA purification and recovery from the lysates were performed using a commercially available system based on the selective binding of nu cleic acids to glass milk. A simple quantitative competitive polymerase cha in reaction (QC-PCR) assay was developed for use in determining copy number s of the internal transcribed spacer (ITS) regions of the ribosomal RNA ope ron (rDNA) in the total DNA extracts. These quantitative analyses demonstra ted that the method using glass bead milling was most effective in recoveri ng 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 e xtract variability. Calculations of average template copy yield per conidiu m in this study indicate that the bead milling method is sufficient to supp ort the detection of less than ten conidia of each of the different organis ms in a PCR assay. (C) 1999 Elsevier Science B.V. All rights reserved.