Abstract:
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.
Keywords: Acquired Immunodeficiency Syndrome/BLOOD/*DIAGNOSIS DNA Polymerases Human HIV-1/GENETICS/*ISOLATION & PURIF Indicators and Reagents Polymerase Chain Reaction/*METHODS RNA-Directed DNA Polymerase RNA, Viral/*BLOOD/GENETICS ABSTRACT 930930
M9391155
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Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
