Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR

Abstract

The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P<0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The Master Pure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.

FIG. 1.

Mean levels of Candida DNA detected in BAL fluid spiked with C. albicans yeast cells and subjected to six DNA extraction methods. Fungal DNA levels were measured using quantitative PCR. Error bars indicate standard deviations for replicate extractions. The MPY and YL-GNOME extraction methods produced the highest levels of Candida DNA.

FIG. 2.

Mean levels of Aspergillus DNA detected in BAL fluid spiked with A. fumigatus conidia and subjected to six DNA extraction methods. Fungal DNA levels were measured using quantitative PCR. Error bars indicate standard deviations for replicate extractions. The UCS and FDNA methods produced the highest levels of Aspergillus DNA from conidia.

FIG. 3.

Mean levels of Aspergillus DNA in tissue culture media inoculated with A. fumigatus conidia, allowed to form mycelia, and subjected to six DNA extraction methods. Fungal DNA levels were measured using quantitative PCR. Error bars indicate standard deviations for replicate extractions. The FDNA method produced the highest levels of Aspergillus DNA from hyphae.