Dickover RE, Donovan RM, Goldstein E, Carlson JR, Bush CE; International Conference on AIDS.

Int Conf AIDS. 1990 Jun 20-23; 6: 161 (abstract no. F.A.342).

University of California, Davis, California, USA

OBJECTIVE: Improved methods for quantitation of the amount of HIV DNA in infected persons are needed for the direct assessment of disease, evaluation of antiviral chemotherapy, and testing of the efficacy of vaccines. We report a methodology for estimating the number of copies of HIV DNA in peripheral blood mononuclear cells (PBMs) using the polymerase chain reaction (PCR). METHODS: PBMs were prepared by centrifugation through LymphoPrep, washed, resuspended in water and autoclaved. The samples were treated with RNase A and Proteinase K, then DNA was extracted using phenol and chloroform. Oligonucleotide pairs from the gag region of HIV (SK38, 39) and the env region (SK 68, 69) were used as primers. The PCR mixture consisted of 1x PCR buffer, four dNTPs at 0.2 mM, 1 muM of each primer, and the test sample adjusted to 1 mug DNA. Thirty reaction cycles were performed. Known amounts of HIV DNA were used to generate a standard curve for the PCR assay. For analysis, the resultant reaction mixtures were blotted onto nitrocellulose, then hybridized with a 32P labeled probe. Hybridized probe was measured by liquid scintillation counting and compared with values from the standard curve. RESULTS: Quantitation of HIV in DNA samples extracted from PBMs from AIDS patients gave a coefficient of variance for the determination of HIV copy number of 14%. Results from 30 patients with AIDS demonstrated that there was a broad range in the number of HIV DNA copies in PBMCs. The number of HIV DNA copies in 1 mug of cellular DNA varied from less than 1,000 to 145,000. The HIV copy number was usually, but not always, stable over time. Changes in the amount of HIV p24 antigen were positively correlated with changes in the number of copies of HIV DNA. CONCLUSION: HIV DNA can be quantified in clinical specimens using the described PCR assay; this test may have clinical use in providing an objective measurement of extent of HIV infection.

Publication Types:

• Meeting Abstracts

Keywords:

• Acquired Immunodeficiency Syndrome
• DNA
• DNA Probes
• Evaluation Studies
• Genes, gag
• HIV
• HIV Antibodies
• HIV Core Protein p24
• HIV Infections
• HIV Long Terminal Repeat
• HIV Seropositivity
• Humans
• Polymerase Chain Reaction
• blood
• genetics
• immunology

Other ID:

• 20034290

UI: 102184248

From Meeting Abstracts