Yeghiazarian T, Wilber J, Kern D, Pachl C, Todd J, Urdea M; International Conference on AIDS.
Int Conf AIDS. 1993 Jun 6-11; 9: 549 (abstract no. PO-B41-2483).
Chiron Corporation, Emeryville, CA.
To determine the relative effect of the sample matrix from different patients on the quantitation of HIV RNA in plasma, HIV-positive plasma collected in EDTA was diluted into ten different HIV-negative plasma samples. Sample dilutions in the linear range were extracted individually, and RT-PCR was performed using the SK 145 and SK 150 primers, followed by liquid hybridization, electrophoresis, and radioanalytic imaging. For the branched DNA (bDNA) signal amplification assay, the samples were centrifuged at 23,500 x g for one hour. The pellet was suspended in a buffer containing proteinase K, detergent, and target oligonucleotide probes and added directly to the wells of a 96-well microplate. Synthetic oligonucleotides hybridize to the pol region of HIV RNA, capturing the RNA onto the surface of a microwell and linking the target to synthetic bDNA amplifier molecules, and then alkaline phosphatase-linked probes hybridize to the immobilized complex. Detection is achieved by incubating the complex with dioxetane and measuring the light emission, and quantitation is determined from a standard curve. The quantitation by RT-PCR of the same sample diluted into different plasma samples ranged from negative to 78 times the expected values, while the bDNA assay ranged from 1.0 to 1.7 times the expected values. Dilution of HIV positive plasma into different negative plasma resulted in varied quantitative values by RT-PCR, including both inhibition and enhancement of signal. This was presumably due to addition of inhibitors of reverse transcriptase and DNA polymerase from some plasma samples and removal of inhibitors by dilution by other plasma samples. On the other hand, the plasma matrix had little effect on quantitation using the bDNA assay.
Publication Types:
Keywords:
- AIDS Vaccines
- Acquired Immunodeficiency Syndrome
- Branched DNA Signal Amplification Assay
- Genes, pol
- HIV
- HIV Infections
- HIV Seropositivity
- Humans
- Oligonucleotide Probes
- RNA
- RNA, Viral
- Reverse Transcriptase Polymerase Chain Reaction
- genetics
- reverse transcriptase, Human immunodeficiency virus 1
Other ID:
UI: 102205511
From Meeting Abstracts