Details:

Alternative Title:
PLoS ONE
Personal Author:
Johnson, Jeffrey A. ; Li, Jin-Fen. ; Wei, Xierong ; Lipscomb, Jonathan. ; Bennett, Diane. ; ... More +
Johnson, Jeffrey A. ; Li, Jin-Fen. ; Wei, Xierong ; Lipscomb, Jonathan. ; Bennett, Diane. ; Brant, Ashley. ; Cong, Mian-er. ; Spira, Thomas. ; Shafer, Robert W. ; Heneine, Walid. ; Less -
Description:
Background The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. Methodology We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. Significance Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.
Subject:
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Anti-HIV Agents
Base Sequence
Biotechnology/Applied Microbiology
Cloning, Molecular
DNA Primers
Drug Resistance, Viral
Gene Amplification
HIV-1
Mutation
Oligodeoxyribonucleotides
Plasmids
Polymerase Chain Reaction
Polymorphism, Genetic
Research Article
Reverse Transcriptase Polymerase Chain Reaction
Sensitivity And Specificity

Source:
PLoS ONE. 2007; 2(7).
Document Type:
Journal Article ;
Collection(s):
CDC Public Access
Main Document Checksum:
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urn:sha256:e03e01a25456f83176c021acc1813a0a0c309954c9ac558f46b56748b9c37c4c
File Type:

Supporting Files

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