Development of a high-throughput detection system for HIV-1 using real-time NASBA based on molecular beacons

Rinie van Beuningen; Salvatore A. Marras; Fred Russell Kramer; Tom Oosterlaken; Jos Weusten; G. Borst; Paul van de Wiel

doi:10.1117/12.424591

16 April 2001 Development of a high-throughput detection system for HIV-1 using real-time NASBA based on molecular beacons

Rinie van Beuningen, Salvatore A. Marras, Fred Russell Kramer, Tom Oosterlaken, Jos Weusten, G. Borst, Paul van de Wiel

Author Affiliations +

Proceedings Volume 4264, Genomics and Proteomics Technologies; (2001) https://doi.org/10.1117/12.424591
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

Abstract

HIV-1 viral load assays require accuracy and sensitivity at low RNA levels with the capability to detect all subtypes. Furthermore, the assay should be easy to perform and fast to be useful for routine diagnostics. In order to meet these demands we have combined isothermal NASBA amplification with molecular beacon probes for real-time detection and quantitation of HIV-1 RNA. Quantitation is based on co-amplification of the HIV-1 RNA in the clinical sample and a synthetic calibrator RNA which is amplified by the same primer set but detected with a differently labeled molecular beacon. The entire procedure is simple and analysis of 48 samples requires less than 1½ hours with minimal hands-on time. A fluorescent plate reader is used for real-time detection and isothermal amplification. The linearity and precision of the assay was determined with the VQC HIV-1 type B standard of the Central Laboratory of the Dutch Red Cross Blood Banks, The Netherlands. Sensitivity was shown to be 50 copies per ml (cps/ml). The average assay precision was 0,19 log10 over a range of 100-300,000 cps/ml tested at nine concentrations. The linearity of dilution series of 15 cultured HIV-1 gag clades A-H was shown. The specificity was 100% on non HIV-1 samples HIV-2, HTLV-1 and HTLV-2. The assay robustness in terms of valid results was 99%. In conclusion, the new real-time NASBA assay meets state-of-the-art HIV-1 viral load performance requirements combined with a high level of user convenience.

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Rinie van Beuningen, Salvatore A. Marras, Fred Russell Kramer, Tom Oosterlaken, Jos Weusten, G. Borst, and Paul van de Wiel "Development of a high-throughput detection system for HIV-1 using real-time NASBA based on molecular beacons", Proc. SPIE 4264, Genomics and Proteomics Technologies, (16 April 2001); https://doi.org/10.1117/12.424591

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