McLaughlin G L, Vodkin M H, Huizinga H W
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana 61801.
J Clin Microbiol. 1991 Feb;29(2):227-30. doi: 10.1128/jcm.29.2.227-230.1991.
By using hybridization at low C0t values, a genomic library on Naegleria fowleri was screened for clones containing repetitive DNA. Partial sequence information from a repetitive clone, Nf9, showed sequence homologies with the mitochondrial ATPase 6 subunit from yeasts and other organisms. Synthetic DNA primers were selected and tested in amplification reactions. Nonstringent hybridization conditions were defined which allowed amplification of N. fowleri DNA and reduced amplification of DNA from nonpathogenic Naegleria species. Stringent conditions were selected which allowed detection only of N. fowleri. Identity of the amplified DNA was confirmed by using internal restriction sites and an internal primer. In a blind study, tissue from mice experimentally infected with N. fowleri was specifically detected by using stringent hybridization conditions.
通过在低Cot值下进行杂交，对福氏耐格里阿米巴的基因组文库进行筛选，以寻找含有重复DNA的克隆。来自重复克隆Nf9的部分序列信息显示，其与酵母和其他生物的线粒体ATP合酶6亚基具有序列同源性。选择并测试了合成DNA引物用于扩增反应。确定了非严格杂交条件，该条件允许扩增福氏耐格里阿米巴的DNA，并减少来自非致病性耐格里阿米巴物种的DNA扩增。选择了严格条件，该条件仅允许检测福氏耐格里阿米巴。通过使用内部限制性酶切位点和内部引物，确认了扩增DNA的同一性。在一项盲法研究中，使用严格杂交条件特异性检测了实验感染福氏耐格里阿米巴的小鼠组织。
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