Mulder J, McKinney N, Christopherson C, Sninsky J, Kwok S; American Society for Microbiology. General Meeting.

Abstr Gen Meet Am Soc Microbiol. 1993; 93: 434 (abstract no. T-15).

Roche Molecular Systems, Alameda, California.

The development of a quantitative assay for particulate viral RNA involves four critical elements: sample preparation, efficient and reproducible amplification of RNA, internal control, and quantitative detection. A single tube, single enzyme system has been developed for quantitation of HIV RNA in plasma or sera by PCR. Reverse transcription and amplification are performed with Tth DNA polymerase, a thermostable enzyme that, under the appropriate conditions, exhibits both reverse transcriptase and DNA polymerase activity. AmpErase (UNG) and dUTP are incorporated into the reactions to ensure that any carryover of DNA from previous amplifications will not compromise quantitation. An internal standard is incorporated into each reaction so that differences in amplification efficiency caused by sample inhibitors, variability in reaction conditions or thermal cycling can be normalized. To insure comparable amplification efficiency, the internal control has the same primer binding regions as the HIV-1 target and generates an amplicon of the same length and base composition. The region flanked by the primers has been shuffled so that the probe binding region is different and thereby can be detected separately. Following amplification, the PCR products are captured and quantified on 96-microwell plates that are coated with either the internal control probe or the HIV-1 probe. For each amplification run, a dilution series of the internal control is amplified and a standard curve generated. The signal of the spiked internal control is compared to the unspiked standard and the signal of the "unknown" HIV-1 product adjusted accordingly. The HIV copy number is determined by extrapolating the "adjusted" HIV signal from the internal control standard curve. Total elapsed time of the assay is less than 8 hours.

Publication Types:

• Meeting Abstracts

Keywords:

• Acquired Immunodeficiency Syndrome
• DNA
• DNA Primers
• DNA Probes
• DNA-Directed DNA Polymerase
• HIV Infections
• HIV-1
• Polymerase Chain Reaction
• RNA
• RNA, Viral
• RNA-Directed DNA Polymerase
• Tth polymerase

Other ID:

• 93291816

UI: 102202539

From Meeting Abstracts