Hrncírová Katerina, Lengerová Martina, Kocmanová Iva, Rácil Zdenek, Volfová Petra, Lochmanová Jana, Dvoráková Dana, Mayer Jirí
Centrum molekulárni biologie a genové terapie, Interní hematoonkologická klinika, FN Brno, Czech Republic.
Klin Mikrobiol Infekc Lek. 2008 Jun;14(3):93-9.
The main goal of this study was to find the best method for Aspergillus DNA isolation from peripheral blood samples. The method should be very effective but not expensive or time consuming to be suitable for routine diagnostics use.
We compared four different methods for Aspergillus DNA isolation - one method with enzymatic lysis of the fungal cell wall and three methods that combine chemical and mechanical lysis (using high speed cell disruption with glass beads). Peripheral blood samples were inoculated with defined amount of Aspergillus fumigatus suspension and used for DNA isolation. Isolated DNA was then quantitatively analyzed with in-house real-time PCR method using specific probe.
Enzymatic method seems not to be useful in a routine diagnostics mainly because of its low efficiency and too long processing time. Better could be the methods using both chemical and mechanical cell disruption that can isolate fungal DNA with high efficiency in a relatively short time.
The method using ZR Fungal/Bacterial DNA Kit (Zymo Research, USA) was chosen for routine use in our laboratory because it is cheap, fast and very efficient.
本研究的主要目标是找到从外周血样本中分离曲霉菌DNA的最佳方法。该方法应非常有效，但成本不高且不耗时，以适用于常规诊断。
我们比较了四种不同的曲霉菌DNA分离方法——一种是酶解真菌细胞壁的方法，另外三种是结合化学和机械裂解的方法（使用玻璃珠进行高速细胞破碎）。将定量的烟曲霉悬浮液接种到外周血样本中，用于DNA分离。然后使用特异性探针通过内部实时PCR方法对分离出的DNA进行定量分析。
酶法在常规诊断中似乎不太有用，主要是因为其效率低且处理时间过长。使用化学和机械细胞破碎的方法可能更好，这些方法可以在相对较短的时间内高效分离真菌DNA。
我们实验室选择使用ZR真菌/细菌DNA试剂盒（美国Zymo Research公司）的方法用于常规检测，因为它便宜、快速且非常有效。
