Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR.
Newer methods such as PCR are being investigated in order to improve the diagnosis of invasive aspergillosis. One of the major obstacles to using PCR to diagnose aspergillosis is a reliable, simple method for extraction of the fungal DNA. The presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of the DNA by conventional methods employed for bacteria. Numerous fungal DNA extraction protocols have been described in the literature. However, these methods are time-consuming, require a high level of skill and may not be suitable for use as a routine diagnostic technique. Here, a number of extraction methods were compared: a freeze-thaw method, a freeze-boil method, enzyme extraction and a bead-beating method using Mini-BeadBeater-8. The quality and quantity of the DNA extracted was compared using real-time PCR. It was found that the use of a bead-beating method followed by extraction with AL buffer (Qiagen) was the most successful extraction technique, giving the greatest yield of DNA, and was also the least time-consuming method assessed.
Griffiths
Lisa J
LJ
Department of Microbiology, Barts and The London NHS Trust, Royal London Hospital, London, UK.
Anyim
Martin
M
Doffman
Sarah R
SR
Department of Respiratory Medicine, Barts and The London NHS Trust, St Bartholomew's Hospital, London.
Wilks
Mark
M
Department of Microbiology, Barts and The London NHS Trust, Royal London Hospital, London, UK.
Millar
Michael R
MR
Department of Microbiology, Barts and The London NHS Trust, Royal London Hospital, London, UK.
Agrawal
Samir G
SG
D001232:Aspergillus fumigatus / Q000235:genetics*; D004271:DNA, Fungal / Q000032:analysis* / Q000302:isolation & purification*; D006801:Humans; D009172:Mycology / Q000379:methods*; D016133:Polymerase Chain Reaction
D004271:DNA, Fungal