Quantification of HIV-1 viral RNA and proviral DNA by isotopic competitive PCR.
A quantitative isotopic competitive PCR (icPCR) assay was established using 32P-labeled primers targeting the HIV-1 gag gene followed by quantification using a phosphoimager. The detection limit varied from 3 to 10 molecules of DNA and 10 to 100 molecules of RNA per reaction. The icPCR quantification of HIV-1 DNA copies correlated well with the cell number of 8E5/LAV cells bearing a single provirus (r2 = 0.95). Provirus quantification was applied to overnight infected donor PBMCs, thereby determining infectious virus titres in culture supernatants as a rapid alternative to limiting dilution culture. Parallel quantification of the HIV-1 RNA indicated the infectious virus fraction to be 0.3%. In 39 HIV-1-infected patients with clinical stages A (n = 17), B (n = 15), and C (n = 7), the HIV-1 RNA in the plasma was determined ranging from 100 to 90600 RNA copies/ml. The results of icPCR and a commercial assay (ROCHE Amplicor HIV-1 Monitor) correlated well (r = 0.97). In 13 additional patients, the plasma viral load per ml was compared with the proviral load per 10(6) PBMC showing a viral excess of 10-1000-fold (mean of 85, r = 0.7, P < 0.01). It is concluded that icPCR is suitable for the measurement of proviral and viral load in experimental and clinical settings.
Gratzl
S
S
Institute for Medical Microbiology, University of Basel Petersplatz 10, Switzerland.
Moroni
C
C
Hirsch
H H
HH
D004279:DNA, Viral / Q000032:analysis*; D015750:Genes, gag / Q000235:genetics; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood; D012680:Sensitivity and Specificity; D019562:Viral Load
D004279:DNA, Viral; D010761:Phosphorus Radioisotopes; D012367:RNA, Viral