Nawrot Urszula, Wlodarczyk Katarzyna, Wrobel Magdalena, Wasik Anita, Dobosz Tadeusz
Department of Microbiology, Wroclaw Medical University, Poland.
Acta Biochim Pol. 2010;57(4):567-71. Epub 2010 Oct 27.
The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (10¹ cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 10² spores and 75 % for 10¹ spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.
本研究的目的是使用选定的商业试剂盒，比较从水中以及接种烟曲霉孢子的血液样本中提取DNA的效率。按照制造商的方案提取DNA之前，先通过酶消化或珠磨法裂解血细胞并破坏真菌细胞。使用针对28S rRNA基因的曲霉特异性引物和SYBR Green I染料或TaqMan探针，通过PCR测定DNA提取效率。所有方法都能在最低测试的孢子水悬浮液密度（10¹个细胞/毫升）下检测到曲霉。使用ZR真菌/细菌DNA试剂盒、YeastStar基因组DNA试剂盒和采用机械细胞破碎的QIAamp DNA Mini试剂盒可获得最高的DNA产量。ZR真菌/细菌DNA试剂盒和YeastStar试剂盒在检测接种曲霉的血液样本时显示出最高的灵敏度（检测10²个孢子时为100%，检测10¹个孢子时为75%）。最近，酶法不再被推荐用于检测血液样本中的曲霉，因此采用机械细胞破碎的ZR真菌/细菌DNA试剂盒和QIAamp DNA Mini试剂盒可用于从临床样本中提取曲霉DNA。
