Coutlee F, Yolken R H, Viscidi R P
Eudowood Division of Infectious Diseases, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
Anal Biochem. 1989 Aug 15;181(1):153-62. doi: 10.1016/0003-2697(89)90410-7.
A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin. Bound DNA-RNA hybrids are detected by an immunoreaction with an enzyme-labeled monoclonal antibody specifically directed against DNA-RNA heteropolymers and the hybrids are quantitatively measured with the addition of a fluorogenic substrate. Optimal conditions under which to perform the assay were hybridization time, 1000 min; temperature, 75 degrees C; probe concentration, 0.2 microgram/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2x SSC. A bisulfite-modified DNA probe was compared to nick-translated probes synthesized with reporter groups of different lengths (bio-11-dUTP or bio-19-dUTP). All probes could detect 10 pg/ml of target RNA. The presence of nonhomologous DNA or RNA sequences reduced the sensitivity of RNA detection by one half-log to 32 pg/ml (1.6 pg/assay).
本文描述并表征了一种用于检测RNA的灵敏非同位素溶液杂交测定法，该方法使用pSP65质粒模型系统。测定过程基于生物素化DNA探针与靶RNA在溶液中的杂交反应。生物素标记的杂交体捕获在包被有抗生物素抗体的微量滴定板上。通过与特异性针对DNA-RNA杂聚物的酶标记单克隆抗体进行免疫反应来检测结合的DNA-RNA杂交体，并通过添加荧光底物对杂交体进行定量测量。进行该测定的最佳条件为：杂交时间1000分钟；温度75℃；探针浓度0.2微克/毫升；探针生物素化程度6.7%；缓冲液严谨性2x SSC。将亚硫酸氢盐修饰的DNA探针与用不同长度报告基团（生物素-11-dUTP或生物素-19-dUTP）合成的缺口平移探针进行了比较。所有探针均可检测到10 pg/ml的靶RNA。非同源DNA或RNA序列的存在使RNA检测的灵敏度降低至32 pg/ml（1.6 pg/测定），降低了半个对数级。
