Quantitation of HIV-1 by real-time amplification and detection

Paul M. Jung; Naiquan Yang; Paul E. Kroeger

doi:10.1117/12.307315

10 April 1998 Quantitation of HIV-1 by real-time amplification and detection

Paul M. Jung, Naiquan Yang, Paul E. Kroeger

Proceedings Volume 3259, Systems and Technologies for Clinical Diagnostics and Drug Discovery; (1998) https://doi.org/10.1117/12.307315
Event: BiOS '98 International Biomedical Optics Symposium, 1998, San Jose, CA, United States

Abstract

A model assay for HIV-1 using a non-competitive internal standard in quantitative RT-PCR was coupled with real-time detection of both analyte and internal standard (IS) signals in a closed system. Real-time detection by the PE-ABI Prism 7700 relied on TaqMan probes specific for HIV and IS. The exogenous, non-competitive IS RNA was added in the same, known amount to a series of HIV RNA standards. The threshold cycle ratio from this internal standard calibration curve was used in the quantitation of HIV. Two configurations of reporter labels were compared. The HEX-HIV:FAM-IS system was the most precise, with nearly half-log discrimination over a range of 10² through 10⁵ copies HIV-1 RNA. The FAM- HIV:HEX-IS system was less precise, but more sensitive and resistant to sample inhibition. The analysis of these signals and their impact on the range and precision of HIV quantitation is discussed. The design and synthesis of the fluorescently-labelled probes is also described.

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Paul M. Jung, Naiquan Yang, and Paul E. Kroeger "Quantitation of HIV-1 by real-time amplification and detection", Proc. SPIE 3259, Systems and Technologies for Clinical Diagnostics and Drug Discovery, (10 April 1998); https://doi.org/10.1117/12.307315

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KEYWORDS

Luminescence

Calibration

Prisms

Signal detection

Sensors

Error analysis

Fluorescence resonance energy transfer

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