IMR Press / FBL / Volume 10 / Issue 1 / DOI: 10.2741/1515

Frontiers in Bioscience-Landmark (FBL) is published by IMR Press from Volume 26 Issue 5 (2021). Previous articles were published by another publisher on a subscription basis, and they are hosted by IMR Press on imrpress.com as a courtesy and upon agreement with Frontiers in Bioscience.

Open Access Article
Quantification of HIV GAG RNA using real time reverse transcriptase PCR
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1 Department of Psychiatry and Behavioral Sciences, University of Miami School of Medicine, Miami, FL 33136, USA
2 Department of Neurology, University of Miami School of Medicine, Miami, FL 33136
3 Department of Pathology, University of Miami School of Medicine, Miami, FL 33136
4 Comprehensive Drug Research Center, University of Miami School of Medicine, Miami, FL 33136
5 Pediatrics McDonald Foundation GeneTeam, University of Miami School of Medicine, Miami, FL 33136
6 Department of Epidemiology, University of Miami School of Medicine, Miami, FL 33136
7 Department of Anthropology, University of Miami, Coral Gables, FL 33124
8 Department of Sociology, University of Miami, Coral Gables, FL 33124

Academic Editor: Alireza Minagar

Front. Biosci. (Landmark Ed) 2005, 10(1), 135–142; https://doi.org/10.2741/1515
Published: 1 January 2005
(This article belongs to the Special Issue Inflammatory disorders of the nervous system)
Abstract

Quantification of HIV-1 is important to quantify risk for disease progression as well as for acquiring infection associated with drug abuse. Prior quantification methods include immune and enzymatic procedures, e.g., quantifying HIV-1 p24 protein by ELISA and the Reverse Transcriptase by enzymatic assay. Improved quantification of HIV-1 RNA and cDNA was established using PCR. This paper describes a real-time PCR technique using the Applied Biosystems 5700 Sequence Detection System and Taqman reverse transcriptase PCR. We initially standardized the PCR method using ribosomal-RNA to obtain relative quantification. Pure gag RNA was used for standard curves, controls, and to obtain absolute RNA quantification. Pure HIV gag RNA was produced by T7-directed transcription of the plasmid pWISP98-85. Detailed statistical analyses describe using absolute standard curves, and intraassay and interassay coefficients of variation to validate the methods. The presented method is highly reproducible and the assay's performance is comparable to prior assays. The assay is validated with an 8-log range down to 80 copies.

Keywords
Real time reverse transcriptase PCR
Taqman
HIV-1
Gag
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