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Journal of Clinical Microbiology, April 2001, p. 1378-1384, Vol. 39, No. 4
Department of Human Retrovirology, Academic
Medical Center, University of Amsterdam,1
PrimaGen,2 and Amsterdam
Institute of Viral Genomics,3 Amsterdam, The
Netherlands
Received 7 September 2000/Returned for modification 6 November
2000/Accepted 26 January 2001
Because human immunodeficiency virus type 1 (HIV-1) subtypes and
circulating recombinant forms (CRFs) are spreading rapidly worldwide
and are becoming less confined to a geographical area, RNA assays that
can detect and quantify all HIV-1 isolates reliably are in demand. We
have developed a fast, real-time monitored RNA assay based on an
isothermal nucleic acid sequence-based amplification technology that
amplifies a part of the long terminal repeat region of the HIV-1
genome. Real-time detection was possible due to the addition of
molecular beacons to the amplification reaction that was monitored in a
fluorimeter with a thermostat. The lower level of detection of the
assay was 10 HIV-1 RNA molecules per reaction, and the lower level of
quantification was 100 copies of HIV-1 RNA with a dynamic range of
linear quantification between 102 and 107 RNA
molecules. All HIV-1 groups, subtypes, and CRFs could be detected and
quantified with equal efficiency, including the group N isolate YBF30
and the group O isolate ANT70. To test the clinical utility of the
assay, a series of 62 serum samples containing viruses that encompassed
subtypes A through G and CRFs AE and AG of HIV-1 group M were analyzed,
and these results were compared to the results of a commercially
available assay. This comparison showed that the quantification results
correlated highly (R2 = 0.735) for those
subtypes that could be well quantified by both assays (subtypes B, C,
D, and F), whereas improved quantification was obtained for subtypes A
and G and CRFs AE and AG. A retrospective study with six individuals
infected with either a subtype A, B, C, or D or an AG isolate of HIV-1
group M, who were treated with highly active antiretroviral therapy,
revealed that the assay was well suited to the monitoring of therapy
effects. In conclusion, the newly developed real-time monitored HIV-1
assay is a fast and sensitive assay with a large dynamic range of
quantification and is suitable for quantification of most if not all
subtypes and groups of HIV-1.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1378-1384.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Single Rapid Real-Time Monitored Isothermal RNA
Amplification Assay for Quantification of Human Immunodeficiency Virus
Type 1 Isolates from Groups M, N, and O
*
Corresponding author. Mailing address: Department of
Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Phone: 31-20-566 6780. Fax: 31-20-691 6531. E-mail:
M.P.deBaar{at}amc.uva.nl.
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