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Science 14 May 1993:
Vol. 260. no. 5110, pp. 976 - 979
DOI: 10.1126/science.8493534
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Articles

Science, Vol 260, Issue 5110, 976-979
Copyright © 1993 by American Association for the Advancement of Science

articles

Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry

BK Patterson, M Till, P Otto, C Goolsby, MR Furtado, LJ McBride, and SM Wolinsky

Department of Pathology, Northwestern University Medical School, Chicago, IL 60611.

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.


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