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Highly sensitive methods based on seminested real-time reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced RNA and proviral DNA. | LitMetric

Highly sensitive methods based on seminested real-time reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced RNA and proviral DNA.

J Clin Microbiol

Department of Medical Microbiology, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam 1105 AZ, The Netherlands.

Published: July 2008

AI Article Synopsis

  • Highly active antiretroviral therapy (HAART) is the main treatment for HIV-1 infection, with effectiveness gauged by measuring viral RNA loads in patients' plasma.
  • While HAART can suppress viral loads to undetectable levels, low-level viral replication continues, risking the emergence of drug-resistant strains.
  • Researchers have developed a new, sensitive method using seminested real-time reverse transcription-PCR (RT-PCR) to accurately quantify HIV-1 RNA and DNA in patient blood samples, improving upon traditional methods.

Article Abstract

The effectiveness of highly active antiretroviral therapy (HAART), the standard of care for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, is assessed by measuring the viral RNA load in plasma. A patient is considered to be successfully treated when the HIV-1 load in plasma stays below the detection limit of commercial assays. However, virus replication and evolution do continue in patients under HAART, which may eventually result in the development of drug-resistant HIV-1 strains and therapy failure. To monitor this low-level virus replication in peripheral blood mononuclear cells (PBMC), sensitive methods are required to measure HIV-1 molecular markers. We report the development of highly sensitive methods for the quantitation of unspliced and multiply spliced HIV-1 RNA and proviral DNA in PBMC. The methods are based on innovative seminested real-time reverse transcription-PCR (RT-PCR) that combines the accuracy and precision of real-time PCR and the sensitivity of nested PCR. We show that the newly developed methods are superior to the conventional single-step real-time RT-PCR in their sensitivity, accuracy, dynamic range, and the power of quantitative detection of HIV-1 RNA and DNA in clinical samples. These easy-to-perform methods can be widely used in research, including clinical studies, to monitor intracellular processes of virus replication.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2446885PMC
http://dx.doi.org/10.1128/JCM.00055-08DOI Listing

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