Comparison of four different methods for extraction of Stachybotrys chartarum spore DNA and verification by real-time PCR.
A comparison of four different methods for the extraction of spore DNA from Stachybotrys chartarum was conducted. Spore DNA was extracted and purified using either one of three different commercial kits or water. All preparations utilized bead milling. Genomic DNA extracted from 10(1) to 10(7) spores was assessed by both agarose gel electrophoresis and real-time quantitative polymerase chain reaction (qPCR) performed against multi-copy (rRNA) and single-(tubulin) gene targets. The spore isolation technique we employed was verified to be pure by light microscopy. Although all preparatory methods led to successful detection by qPCR, S. chartarum spore DNA prepared using the Qiagen Plant kit was notably better over the extraction range.
Black
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JA
RTI International, 3040 Cornwallis Road, Research Triangle Park, NC 27709, USA. jab@rti.org
Foarde
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KK
D004271:DNA, Fungal / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D016681:Genome, Fungal; D008967:Molecular Biology / Q000379:methods*; D016133:Polymerase Chain Reaction; D013172:Spores, Fungal / Q000737:chemistry* / Q000235:genetics; D013191:Stachybotrys / Q000737:chemistry* / Q000235:genetics; D014404:Tubulin / Q000235:genetics
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D014404:Tubulin