Fredricks David N, Smith Caitlin, Meier Amalia
Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
J Clin Microbiol. 2005 Oct;43(10):5122-8. doi: 10.1128/JCM.43.10.5122-5128.2005.
The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P<0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The Master Pure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.
通过聚合酶链反应（PCR）检测临床样本中的真菌病原体，需要使用能够有效裂解真菌细胞并回收适合扩增的DNA的提取方法。我们使用定量PCR测定法来测量两种重要真菌病原体在六种DNA提取方法下的DNA回收率。将烟曲霉分生孢子或白色念珠菌酵母细胞添加到支气管肺泡灌洗液中，然后进行DNA提取，以评估从一定数量的真菌繁殖体中回收DNA的情况。为了模拟组织中的菌丝生长，让烟曲霉分生孢子在组织培养基中形成菌丝体，然后收获用于DNA提取。在三个实验系统中的每一个中，六种提取方法的DNA产量差异都非常显著（P<0.0001）。一种基于真菌细胞壁酶解的提取方法（酵母细胞裂解加上使用GNOME试剂盒）对白色念珠菌产生了高水平的真菌DNA，但对烟曲霉分生孢子或菌丝体产生的真菌DNA水平较低。采用珠子机械搅拌的提取方法对烟曲霉菌丝体产生的产量最高。Master Pure酵母方法从白色念珠菌中产生了高水平的DNA，但从烟曲霉中产生的产量仅为中等。一种提取方法中的一种试剂被真菌DNA污染，包括来自曲霉属和念珠菌属的DNA。总之，这六种提取方法产生的真菌DNA产量明显不同，因此会显著影响真菌PCR检测的结果。没有一种提取方法对所有生物体都是最佳的。
