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Journal of Clinical Microbiology, July 2008, p. 2206-2211, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.00055-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Alexander O. Pasternak, Karen W. Adema, Margreet Bakker, Suzanne Jurriaans, Ben Berkhout, Marion Cornelissen, and Vladimir V. Lukashov*

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Received 11 January 2008/ Returned for modification 27 February 2008/ Accepted 30 April 2008

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.

* Corresponding author. Mailing address: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam 1105 AZ, The Netherlands. Phone: 31-20-5665861. Fax: 31-20-5669064. E-mail: v.lukashov{at}amc.uva.nl

{triangledown} Published ahead of print on 7 May 2008.

Journal of Clinical Microbiology, July 2008, p. 2206-2211, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.00055-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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