Summary
An improved Alu-long terminal repeat (LTR) polymerase chain reaction (PCR) assay is described for the quantification of integrated HIV-1 DNA in infected cells. The method includes generation of an infected cell line containing numerous randomly distributed HIV-1 integrated DNA for the construction of the DNA standard and a two-step realtime PCR assay in which the first-round PCR amplifies the DNA sequence between the HIV-1 LTR and the nearest chromosomal Alu element, and the nested PCR specifically amplifies PCR products from the first-round PCR. This assay allows us to quantify proviral DNA with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents) and exhibits a broad range of quantification spanning 5 log10 provirus copies. This Alu-LTR-based real-time nested PCR assay may be particularly useful to quantify integrated HIV-1 DNA in patients. It may also allow for the precise study of integration of HIV-1 DNA or HIV-1 based lentiviral vectors and may be a valuable tool to test future inhibitors of integration.
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Acknowledgments
A. Brussel was supported by fellowships from the Agence Nationale de Recherche sur le SIDA and from the Fondation pour la Recherche Médicale. We thank U. Hazan for providing the pR7 Neo Δenv and pVSV-G vectors and for constant advice in the development of the integration assay. We acknowledge Olfert Landt (TIB MOLBIOL) for excellent technical assistance with the design of primers and hybridization probes. We would also like to thank C. Petit, S. Pierre, M. Alizon, and B. Canque for helpful discussions.
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Brussel, A., Delelis, O., Sonigo, P. (2005). Alu-LTR Real-Time Nested PCR Assay for Quantifying Integrated HIV-1 DNA. In: Zhu, T. (eds) Human Retrovirus Protocols. Methods in Molecular Biology™, vol 304. Humana Press. https://doi.org/10.1385/1-59259-907-9:139
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DOI: https://doi.org/10.1385/1-59259-907-9:139
Publisher Name: Humana Press
Print ISBN: 978-1-58829-495-1
Online ISBN: 978-1-59259-907-3
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