Mulder J, McKinney N, Christopherson C, Kwok S; International Conference on AIDS.

Int Conf AIDS. 1993 Jun 6-11; 9: 264 (abstract no. PO-A32-0777).

Roche Molecular Systems, Alameda, CA.

The quantitation of HIV plasma viremia may prove useful in monitoring disease progression, responsiveness of patients to a therapeutic regimen, and in determining efficacy of therapeutic vaccines. We have developed a single tube, single enzyme system for quantitation of HIV RNA in plasma by PCR. Reverse transcription and amplification are performed with Tth DNA polymerase, a thermostable enzyme that, under appropriate conditions, exhibits both reverse transcriptase and DNA polymerse activity. Uracil-N-glycosylase (UNG) and dUTP are incorporated into the reactions to avoid problems associated with PCR product carryover. An internal standard RNA transcript that has the same primer binding regions as the HIV-1 target and generates an amplicon of the same length and base composition but contains a different probe binding region has been constructed. The internal control is spiked into each reaction and is used to normalize differences in amplification efficiency caused by sample interferents, variability in reaction conditions or thermal cycling. PCR products are captured and quantified on 96-microwell plates that are coated with either the internal control probe or the HIV-1 probe. This colorimetric microwell assay format is similar to that currently used for the HIV-1 qualitative assay but has been modified to provide a three log dynamic range for detection. For each PCR run, a dilution series of the internal control is amplified and a standard curve generated. The signal of each spiked internal control is compared to the unspiked standard and any difference is used to adjust the signal of the "unknown" HIV-1 product. The HIV copy number is determined by extrapolating the "adjusted" HIV signal from the standard curve. The assay is highly reproducible and sensitive.

Publication Types:

• Meeting Abstracts

Keywords:

• AIDS Vaccines
• Acquired Immunodeficiency Syndrome
• Anti-HIV Agents
• DNA
• DNA Glycosylases
• DNA Primers
• DNA-Directed DNA Polymerase
• HIV
• HIV Infections
• HIV Long-Term Survivors
• HIV Seropositivity
• HIV-1
• Humans
• Polymerase Chain Reaction
• RNA
• RNA-Directed DNA Polymerase
• Tth polymerase
• Viremia

Other ID:

• 93334264

UI: 102203638

From Meeting Abstracts