An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR.
To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene.
Karakousis
A
A
Queen Elizabeth Hospital, Department of ENT Surgery and the Department of Surgery, The University of Adelaide, Woodville Road, Woodville South, South Australia, 5011, Australia.
Tan
L
L
Ellis
D
D
Alexiou
H
H
Wormald
P J
PJ
D002473:Cell Wall / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D005658:Fungi / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics
D004271:DNA, Fungal; D012337:RNA, Ribosomal, 18S