[Frontiers in Bioscience 10, 135-142, January 1, 2005]

QUANTIFICATION OF HIV GAG RNA USING REAL TIME REVERSE TRANSCRIPTASE PCR

Paul Shapshak 1-5, Robert Duncan 6, Clyde B. McCoy 4, 6 and J. Bryan Page 1, 4, 7, 8

Departments of 1 Psychiatry and Behavioral Sciences, 2 Neurology, 3 Pathology, 4 Comprehensive Drug Research Center, 5 Pediatrics McDonald Foundation GeneTeam, 6 Epidemiology, University of Miami School of Medicine, Miami, FL 33136, Department of 7 Anthropology, University of Miami, Coral Gables, FL 33124, Department of 8 Sociology, University of Miami, Coral Gables, FL 33124

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Methods
3.1. Description and Preparation of Plasmid pWIS98-85
3.2. RNA transcription from pWIS98-85
3.3. RNA extraction
3.4. Real Time PCR
3.5. Statistics
4. Results
5. Discussion
6. Acknowledgments
7. References

1. 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.