Karakousis A, Tan L, Ellis D, Alexiou H, Wormald P J
Queen Elizabeth Hospital, Department of ENT Surgery and the Department of Surgery, The University of Adelaide, Woodville Road, Woodville South, South Australia, 5011, Australia.
J Microbiol Methods. 2006 Apr;65(1):38-48. doi: 10.1016/j.mimet.2005.06.008. Epub 2005 Aug 15.
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.
迄今为止，尚无一种已报道的DNA提取方法适用于从所有真菌物种中高效提取DNA。在基于PCR的医学诊断应用中，提取效率尤为重要，因为组织活检中真菌的数量可能有限。我们对16种医学相关真菌采用了物理、化学和酶促细胞壁破坏方法，这是提取DNA的第一步。光学显微镜检查表明，用研钵和研杵研磨是破坏菌丝和分生孢子坚硬真菌细胞壁的最有效方法。然后，我们试验了几种已发表的DNA分离方案，以确定最有效的提取方法。通过加入溶菌酶和蛋白酶K酶促消化步骤，并改编自商业试剂盒（MO BIO）的DNA提取程序，从所有16个物种中获得了高质量DNA的高产量，从而实现了最佳提取。通过成功PCR扩增真菌18S小亚基rRNA多拷贝基因的保守区域，证实了DNA质量。
