Patterson B K, Till M, Otto P, Goolsby C, Furtado M R, McBride L J, Wolinsky S M
Department of Pathology, Northwestern University Medical School, Chicago, IL 60611.
Science. 1993 May 14;260(5110):976-9. doi: 10.1126/science.8493534.
Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.
通过聚合酶链式反应扩增直接取自HIV-1感染患者的细胞系和血液中的1型人类免疫缺陷病毒（HIV-1）DNA及信使核糖核酸序列，并与荧光素标记的探针进行原位杂交，然后通过流式细胞术分析单个标记的细胞。经过流式细胞术分析，异质性细胞群体可重复性地解析为HIV-1阳性和阴性分布。荧光显微镜检查显示细胞形态得以保留，扩增产物DNA的细胞内定位得以维持。未观察到非特异性探针的滞留现象。对HIV-1感染患者血液中的细胞进行前病毒DNA和病毒信使核糖核酸分析表明，HIV-1基因组存在于大量潜伏感染细胞的储存库中。使用这项技术，现在能够以单细胞分辨率在悬浮细胞的一个小亚群中快速且可重复地检测单拷贝DNA或低丰度信使核糖核酸，并对这些细胞进行分选以进一步鉴定。
