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Detection of HIV-1 DNA and Messenger RNA in Individual Cells by PCR-Driven in Situ Hybridization and Flow Cytometry
Abstract
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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Citations
Cited by
Viral dynamics in human immunodeficiency virus type 1 infection
Wei, X., Ghosh, S. K., Taylor, M. E., Johnson, V. A., Emini, E. A., Deutsch, P., Lifson, J. D., Bonhoeffer, S., Nowak, M. A., Hahn, B. H., et al. (1995) · Nature · doi:10.1038/373117a0 · Cited by 3,260
Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection
Chun, T.-W., Carruth, L., Finzi, D., Shen, X., DiGiuseppe, J. A., Taylor, H., Hermankova, M., Chadwick, K., Margolick, J., Quinn, T. C., et al. (1997) · Nature · doi:10.1038/387183a0 · Cited by 2,050
In vivo fate of HIV-1-infected T cells: Quantitative analysis of the transition to stable latency
Chun, T.-W., Finzi, D., Margolick, J., Chadwick, K., Schwartz, D., & Siliciano, R. F. (1995) · Nature Medicine · doi:10.1038/nm1295-1284 · Cited by 787
Quantitative Image Analysis of HIV-1 Infection in Lymphoid Tissue
Haase, A. T., Henry, K., Zupancic, M., Sedgewick, G., Faust, R. A., Melroe, H., Cavert, W., Gebhard, K., Staskus, K., Zhang, Z.-Q., et al. (1996) · Science · doi:10.1126/science.274.5289.985 · Cited by 599
Highly multiplexed simultaneous detection of RNAs and proteins in single cells
Frei, A. P., Bava, F.-A., Zunder, E. R., Hsieh, E. W. Y., Chen, S.-Y., Nolan, G. P., & Gherardini, P. F. (2016) · Nature Methods · doi:10.1038/nmeth.3742 · Cited by 330
Metadata
- Authors
- Patterson, B. K., Till, M., Otto, P., Goolsby, C., Furtado, M. R., McBride, L. J., & Wolinsky, S. M.
- Venue
- Science
- Published
- 1993-05-14
- Volume
- 260
- Issue
- 5110
- Pages
- 976-979
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- OpenAlex
- W2020554721
- Cited by
- 264
- References
- 0
- FWCI
- 11.79
- Type
- journal article
- Language
- en
- Open access
- closed
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- Patterson, B. K., Till, M., Otto, P., Goolsby, C., Furtado, M. R., McBride, L. J., & Wolinsky, S. M. (1993). Detection of HIV-1 DNA and Messenger RNA in Individual Cells by PCR-Driven in Situ Hybridization and Flow Cytometry. Science. https://doi.org/10.1126/science.8493534
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