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Journal of Clinical Microbiology, April 2001, p. 1378-1384, Vol. 39, No. 4
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

Michel P. de Baar,1,* Maaike W. van Dooren,2 Esther de Rooij,1,2 Margreet Bakker,1,3 Bob van Gemen,2 Jaap Goudsmit,1,3 and Anthony de Ronde2

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.

* 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.

Journal of Clinical Microbiology, April 2001, p. 1378-1384, Vol. 39, No. 4
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.4.1378-1384.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.