DETECTION OF HIV-1 DNA AND MESSENGER-RNA IN INDIVIDUAL CELLS BY PCR-DRIVEN INSITU HYBRIDIZATION AND FLOW-CYTOMETRY
DETECTION OF HIV-1 DNA AND MESSENGER-RNA IN INDIVIDUAL CELLS BY PCR-DRIVEN INSITU HYBRIDIZATION AND FLOW-CYTOMETRY
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DOI：
10.1126/science.8493534
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发表时间：
1993-05-14
期刊：
影响因子：
56.9
通讯作者：
WOLINSKY, SM
中科院分区：
文献类型：
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作者：
PATTERSON, BK;TILL, M;WOLINSKY, SM
人类免疫缺陷病毒1型（HIV-1）的DNA和信使RNA序列在两个细胞系和血液中直接从HIV-1感染的患者进行了扩增，通过聚合酶链反应和杂交荧光标记的探针原位，并单独标记的细胞进行了流式细胞术分析。流式细胞仪分析后，异质性细胞群可重复地分解为HIV-1阳性和阴性分布。荧光显微镜观察显示，细胞形态保持不变，扩增产物DNA的胞内定位得以维持。未观察到非特异性探针的保留。对HIV-1感染者血液细胞中的前病毒DNA和病毒信使RNA的分析表明，HIV-1基因组存在于大量潜伏感染细胞中。通过使用这种技术，现在可以以单细胞分辨率在悬浮液中的细胞的少数亚群中快速且可重复地检测单拷贝DNA或低丰度信使RNA，并对这些细胞进行分选以进一步表征。
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.