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Quantification of HIV-1 viral RNA and proviral DNA by isotopic competitive PCR

Gratzl, S. and Moroni, C. and Hirsch, H. H.. (1997) Quantification of HIV-1 viral RNA and proviral DNA by isotopic competitive PCR. Journal of Virological Methods, Vol. 66, H. 2. pp. 269-282.

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Official URL: http://edoc.unibas.ch/dok/A5257889

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Abstract

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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Growth and Development (Moroni)
03 Faculty of Medicine > Departement Biomedizin > Former Units at DBM > Growth and Development (Moroni)
UniBasel Contributors:Moroni, Christoph
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Elsevier
ISSN:0166-0934
Note:Publication type according to Uni Basel Research Database: Journal article
Last Modified:22 Mar 2012 14:20
Deposited On:22 Mar 2012 13:19

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