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A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load

Nature Protocols volume 3, pages 1240–1248 (2008)Cite this article

Abstract

Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1–infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4+ T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.

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Figure 1
Figure 2
Figure 3: Amplification plots obtained with the HIV-1 plasmid control (shown in red), uninfected human genomic DNA (hDNA; green) and a no-template control (NTC; blue).
Figure 4: Standard curves for the CCR5 and the HIV-1 real-time PCR assays.
Figure 5: Effects of HIV-1 genetic variability on HIV-1 load quantification.

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Acknowledgements

This work was supported by the WHO-UNAIDS Network for HIV Isolation and Characterization, which provided primary HIV-1 isolates through the WHO Repository at the Centre for AIDS reagents (CFAR), National Institute for Biological Standard and Control (NIBSC), South Mimms, UK, and by grants from the VI National AIDS Project, Istituto Superiore di Sanitá, Rome, Italy.

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Author notes

  1. Paolo Lusso

    Present address: Present address: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA.,

  2. Mauro S Malnati and Gabriella Scarlatti: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological and Technological Research (DIBIT), Unit of Human Virology, San Raffaele Scientific Institute, Via Olgettina n. 58, Milan, 20132, Italy

    Mauro S Malnati, Francesca Gatto, Giulia Cassina, Teresa Rutigliano & Paolo Lusso

  2. Department of Biological and Technological Research (DIBIT), Unit of Viral Evolution and Transmission, San Raffaele Scientific Institute, Via Olgettina n. 58, Milan, 20132, Italy

    Gabriella Scarlatti, Francesca Salvatori & Rosy Volpi

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Correspondence to Mauro S Malnati or Paolo Lusso.

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Malnati, M., Scarlatti, G., Gatto, F. et al. A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load. Nat Protoc 3, 1240–1248 (2008). https://doi.org/10.1038/nprot.2008.108

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