Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (190K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Weusten, J. J. A. M.
Right arrow Articles by van de Wiel, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Weusten, J. J. A. M.
Right arrow Articles by van de Wiel, P. A.
Related Collections
Right arrow Nucleic acid amplification
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2002, Vol. 30, No. 6 e26
© 2002 Oxford University Press

Principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons

Jos J. A. M. Weusten*, Wim M. Carpay, Tom A. M. Oosterlaken, Martien C. A. van Zuijlen1 and Paul A. van de Wiel

bioMérieux, Nucleic Acid Diagnostics, Boseind 15, PO Box 84, 5280 AB Boxtel, The Netherlands and 1Department of Mathematics, Catholic University Nijmegen, Nijmegen, The Netherlands

For quantitative NASBA-based viral load assays using homogeneous detection with molecular beacons, such as the NucliSens EasyQ HIV-1 assay, a quantitation algorithm is required. During the amplification process there is a constant growth in the concentration of amplicons to which the beacon can bind while generating a fluorescence signal. The overall fluorescence curve contains kinetic information on both amplicon formation and beacon binding, but only the former is relevant for quantitation. In the current paper, mathematical modeling of the relevant processes is used to develop an equation describing the fluorescence curve as a function of the amplification time and the relevant kinetic parameters. This equation allows reconstruction of RNA formation, which is characterized by an exponential increase in concentrations as long as the primer concentrations are not rate limiting and by linear growth over time after the primer pool is depleted. During the linear growth phase, the actual quantitation is based on assessing the amplicon formation rate from the viral RNA relative to that from a fixed amount of calibrator RNA. The quantitation procedure has been successfully applied in the NucliSens EasyQ HIV-1 assay.

* To whom correspondence should be addressed. Tel: +31 411 654 509; Fax: +31 411 654 311; Email: jos.weusten{at}eu.biomerieux.com


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
J. J. Li, Y. Chu, B. Y.-H. Lee, and X. S. Xie
Enzymatic signal amplification of molecular beacons for sensitive DNA detection
Nucleic Acids Res., April 1, 2008; 36(6): e36 - e36.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Microbiol.Home page
W. Stevens, P. Horsfield, and L. E. Scott
Evaluation of the Performance of the Automated NucliSENS easyMAG and EasyQ Systems versus the Roche AmpliPrep-AMPLICOR Combination for High-Throughput Monitoring of Human Immunodeficiency Virus Load
J. Clin. Microbiol., April 1, 2007; 45(4): 1244 - 1249.
[Abstract] [Full Text] [PDF]

Home page
 J Antimicrob ChemotherHome page
B. Rodes, J. Sheldon, C. Toro, V. Jimenez, M. A. Alvarez, and V. Soriano
Susceptibility to protease inhibitors in HIV-2 primary isolates from patients failing antiretroviral therapy
J. Antimicrob. Chemother., April 1, 2006; 57(4): 709 - 713.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
E. T. Casper, J. H. Paul, M. C. Smith, and M. Gray
Detection and Quantification of the Red Tide Dinoflagellate Karenia brevis by Real-Time Nucleic Acid Sequence-Based Amplification
Appl. Envir. Microbiol., August 1, 2004; 70(8): 4727 - 4732.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. J. A. M. Weusten, P. A. W. M. Wouters, M. C. A. van Zuijlen, and P. A. van de Wiel
Stochastic processes defining sensitivity and variability of internally calibrated quantitative NASBA-based viral load assays
Nucleic Acids Res., December 15, 2002; 30(24): e137 - e137.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.