Van de Wiel P, Top B, Weusten J, Oosterlaken T; International Conference on AIDS.
Int Conf AIDS. 2000 Jul 9-14; 13: abstract no. MoPeA2125.
P. Van de Wiel, Organon Teknika BV, Dept. Nucleic Acid Diagnostics, Boseind 15, NL-5281 RM Boxtel, Netherlands, Tel.: +31 411 654 316, Fax: +31 411 654 311, E-mail: p.vandewiel@teknika.btl.akzonobel.nl
A new assay for quantification of HIV-1 RNA was developed by combining the NucliSens system with homogeneous molecular beacon detection. Molecular beacons are nucleic acid probes with a quenched fluorophore that undergo a conformational change upon binding to a target sequence. This results in an increase in fluorescence. Plasma samples are added to lysis buffer, thus causing disintegration of the virus and release of nucleic acid. Internal calibrator RNA is added to the sample and co-extracted with HIV-1 RNA using the NucliSens Extractor. Subsequently, the isolated HIV-1 and calibrator RNA are amplified in a NASBA reaction using gag primers. A molecular beacons complementary to a highly conserved region of the HIV-1 genome and a beacon with a different label complementary to the calibrator RNA are added before amplification. Amplification at 41-C and real-time detection is performed in a NucliSens fluorescence reader. The HIV-1 RNA concentration in the sample and validity of the test result is calculated by comparing fluorescence kinetics of the specific HIV-1 beacon and the calibrator beacon. With this assay 48 samples could be amplified and detected within 90 minutes, including 30 minutes hands-on time. HIV-1 RNA could be accurately measured in samples containing an HIV-1 RNA subtype B reference standard in concentrations ranging from 50 up to 300,000 copies/ml. Linearity and parallelism of dilutions of culture supernatants of HIV-1 clades A-H of group M was demonstrated. Testing of clinical samples with a suspected high percentage non-B clades showed an excellent subtype reactivity of the real-time beacon assay compared to the current NucliSens HIV-1 QT utilizing electrochemiluminescence detection. In conclusion, data show that the new NucliSens assay is the first real-time amplification assay for HIV-1 viral load measurement combining state-of-the-art diagnostic quality, high throughput and a high level of user convenience.
Publication Types:
Keywords:
- Base Sequence
- Biological Assay
- CD4 Lymphocyte Count
- Genes, gag
- HIV-1
- Laboratory Techniques and Procedures
- RNA
- RNA, Viral
- Self-Sustained Sequence Replication
- analysis
- genetics
- organization & administration
Other ID:
UI: 102237672
From Meeting Abstracts