Simple PCR Assays Improve the Sensitivity of HIV-1 Subtype B Drug Resistance Testing and Allow Linking of Resistance Mutations

Details

• Alternative Title:
  PLoS ONE
• Personal Author:
  Johnson, Jeffrey A. ; Li, Jin-Fen. ; Wei, Xierong ; Lipscomb, Jonathan. ; Bennett, Diane. ; Brant, Ashley. ; Cong, Mian-er. ; Spira, Thomas. ; Shafer, Robert W. ; Heneine, Walid.
• 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.
  
  
• Subjects:
  Anti-HIV Agents Base Sequence Cloning, Molecular DNA Primers Drug Resistance, Viral Gene Amplification HIV-1 Mutation Oligodeoxyribonucleotides Plasmids Polymerase Chain Reaction Polymorphism, Genetic Reverse Transcriptase Polymerase Chain Reaction Sensitivity And Specificity

  More +
• Source:
  PLoS ONE. 2007; 2(7).
• Document Type:
  Journal Article
• Volume:
  2
• Issue:
  7
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha-512:b40c833d047ccb39ee875cd15c03cf68d29fb277781c274d07498b6663dc24bd8956351b5a739fdddbd35cc936b0dcdb4db19d2ff4c4b0495e34f80c9a929a6f
• Download URL:
  https://stacks.cdc.gov/view/cdc/4252/cdc_4252_DS1.pdf
• File Type:
  [PDF - 321.52 KB ]