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Journal of Clinical Microbiology, July 2009, p. 2194-2199, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.01264-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development and Assessment of a Multiplex Real-Time PCR Assay for Quantification of Human Immunodeficiency Virus Type 1 DNA{triangledown}

A. Beloukas,{dagger} D. Paraskevis,{dagger} C. Haida, V. Sypsa, and A. Hatzakis*

Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece

Received 4 July 2008/ Returned for modification 17 January 2009/ Accepted 27 April 2009

Previous studies showed that high levels of human immunodeficiency virus type 1 (HIV-1) DNA are associated with a faster progression to AIDS, an increased risk of death, and a higher risk of HIV RNA rebound in patients on highly active antiretroviral therapy. Our objective was to develop and assess a highly sensitive real-time multiplex PCR assay for the quantification of HIV-1 DNA (RTMP-HIV) based on molecular beacons. HIV-1 DNA quantification was carried out by RTMP in a LightCycler 2.0 apparatus. HIV-1 DNA was quantified in parallel with CCR5 as a reference gene, and reported values are numbers of HIV-1 DNA copies/106 peripheral blood mononuclear cells (PBMCs). The clinical sensitivity of the assay was assessed for 115 newly diagnosed HIV-1-infected individuals. The analytical sensitivity was estimated to be 12.5 copies of HIV-1 DNA per 106 PBMCs, while the clinical sensitivity was 100%, with levels ranging from 1.23 to 4.25 log10 HIV-1 DNA copies/106 PBMCs. In conclusion, we developed and assessed a new RTMP-HIV assay based on molecular beacons, using a LightCycler 2.0 instrument. This multiplex assay has comparable sensitivity, reproducibility, and accuracy to single real-time PCR assays.

* Corresponding author. Mailing address: Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, M. Asias 75, GR-115 27 Athens, Greece. Phone: (30210) 7462090. Fax: (30210) 7462190. E-mail: ahatzak{at}med.uoa.gr

{triangledown} Published ahead of print on 6 May 2009.

{dagger} The first two authors contributed equally to this work.

Journal of Clinical Microbiology, July 2009, p. 2194-2199, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.01264-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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