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Journal of Clinical Microbiology, May 2001, p. 1895-1902, Vol. 39, No. 5
Department of Human Retrovirology, Academic
Medical Center, University of Amsterdam,1
PrimaGen,2 and Amsterdam
Institute of Viral Genomics,3 Amsterdam, The
Netherlands
Received 16 October 2000/Returned for modification 6 December
2000/Accepted 23 February 2001
To halt the human immunodeficiency virus type 1 (HIV-1) epidemic
requires interventions that can prevent transmission of numerous HIV-1
subtypes. The most frequently transmitted viruses belong to the
subtypes A, B, and C and the circulating recombinant forms (CRFs) AE
and AG. A fast one-tube assay that identifies and distinguishes among
subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The
assay amplifies a part of the gag gene sequence of the
genome of all currently known HIV-1 subtypes and can identify and
distinguish among the targeted subtypes as the reaction proceeds,
because of the addition of subtype-specific molecular beacons with
multiple fluorophores. The combination of isothermal nucleic acid
sequence-based amplification and molecular beacons is a new approach in
the design of real-time assays. To obtain a sufficiently specific
assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype
A and CRF AG isolates reacted with the same molecular beacon. We tested
the specificity and sensitivity of the assay on a panel of the culture
supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A
through G, CRF AE and AG, a group O isolate, and a group N isolate.
Assay sensitivity on this panel was 92%, with 89% correct subtype
identification relative to sequence analysis. A linear relationship was
found between the amount of input RNA in the reaction mixture and the
time that the reaction became positive. The lower detection level of
the assay was approximately 103 copies of HIV-1 RNA per
reaction. In 38% of 50 serum samples from HIV-1-infected individuals
with a detectable amount of virus, we could identify subtype sequences
with a specificity of 94% by using sequencing and phylogenetic
analysis as the "gold standard." In conclusion, we showed the
feasibility of the approach of using multiple molecular beacons labeled
with different fluorophores in combination with isothermal
amplification to identify and distinguish subtypes A, B, and C and CRFs
AE and AG of HIV-1. Because of the low sensitivity, the assay in this
format would not be suited for clinical use but can possibly be used
for epidemiological monitoring as well as vaccine research studies.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1895-1902.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
One-Tube Real-Time Isothermal Amplification Assay To Identify
and Distinguish Human Immunodeficiency Virus Type 1 Subtypes A, B,
and C and Circulating Recombinant Forms AE and AG
*
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, May 2001, p. 1895-1902, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1895-1902.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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