Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR.
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.
Fredricks
David N
DN
Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA. dfredric@fhcrc.org
Smith
Caitlin
C
Meier
Amalia
A
D001228:Aspergillosis / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D002176:Candida albicans / Q000235:genetics / Q000302:isolation & purification*; D002177:Candidiasis / Q000382:microbiology; D004271:DNA, Fungal / Q000032:analysis / Q000302:isolation & purification*; D004866:Equipment Contamination; D006801:Humans; D008172:Lung Diseases, Fungal / Q000175:diagnosis* / Q000382:microbiology*; D011014:Pneumonia / Q000175:diagnosis / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*
D004271:DNA, Fungal