Quantification of HIV GAG RNA using real time reverse transcriptase PCR.
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.
Shapshak
Paul
P
Department of Psychiatry and Behavioral Sciences, University of Miami School of Medicine, Miami, FL 33136, USA. pshapsha@med.miami.edu <pshapsha@med.miami.edu>
Duncan
Robert
R
McCoy
Clyde B
CB
Page
J Bryan
JB
D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D015233:Models, Statistical; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000737:chemistry; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014764:Viral Proteins / Q000737:chemistry
D015683:Gene Products, gag; D012335:RNA, Ribosomal; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases