Steffan R T, Atlas R M
University of Louisville.
Biotechniques. 1990 Mar;8(3):316-8.
A solution hybridization method was developed for detecting genetically engineered microorganisms in environmental samples. The detection method involves recovery of DNA from the microbial community of an environmental sample followed by hybridization in solution with a radiolabeled RNA gene probe. After nuclease digestion of non-hybridized probe RNA, the DNA-RNA hybrids formed in the solution hybridization reaction are separated by sephadex or hydroxyapatite column chromatography and detected by liquid scintillation counting. Using solution hybridization-gene probe detection, as few as 100-1000 target cells per gram sediment sample of a 2,4,5-T-degrading genetically engineered microorganisms could be detected.
开发了一种溶液杂交方法,用于检测环境样品中的基因工程微生物。该检测方法包括从环境样品的微生物群落中回收DNA,然后与放射性标记的RNA基因探针在溶液中进行杂交。在对未杂交的探针RNA进行核酸酶消化后,溶液杂交反应中形成的DNA-RNA杂交体通过葡聚糖凝胶或羟基磷灰石柱色谱法分离,并通过液体闪烁计数进行检测。使用溶液杂交-基因探针检测,每克含有降解2,4,5-T的基因工程微生物的沉积物样品中低至100 - 1000个靶细胞都能被检测到。