DETECTION OF HIV-1 DNA AND MESSENGER-RNA IN INDIVIDUAL CELLS BY PCR-DRIVEN INSITU HYBRIDIZATION AND FLOW-CYTOMETRY

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequ ences in both cell lines and blood obtained directly from HIV-1-infect ed patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled c ells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-p ositive and -negative distributions. Fluorescence microscopy showed th at the cellular morphology was preserved and intracellular localizatio n of amplified product DNA was maintained. Retention of nonspecific pr obe 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. Wit h the use of this technique it is now possible to detect single-copy D NA 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.