Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay

J Clin Microbiol. 2001 Apr;39(4):1303-10. doi: 10.1128/JCM.39.4.1303-1310.2001.

Abstract

Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log(10). Reproducibility was evaluated in intra- and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 10(6) PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 10(6) PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy (P = 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5' end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4(+) cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-HIV Agents / therapeutic use
  • Base Sequence
  • DNA, Viral / blood*
  • Drug Therapy, Combination
  • Ganglia / virology
  • HIV Infections / drug therapy
  • HIV Infections / virology*
  • HIV-1 / genetics
  • HIV-1 / physiology*
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Proviruses*
  • RNA, Viral / blood
  • Rectum / virology
  • Reproducibility of Results
  • Reverse Transcriptase Inhibitors / therapeutic use
  • Sensitivity and Specificity
  • Taq Polymerase / metabolism
  • Viral Load*

Substances

  • Anti-HIV Agents
  • DNA, Viral
  • RNA, Viral
  • Reverse Transcriptase Inhibitors
  • Taq Polymerase

Associated data

  • GENBANK/AF277299
  • GENBANK/AF277300
  • GENBANK/AF277301
  • GENBANK/AF277302
  • GENBANK/AF277303
  • GENBANK/AF277304
  • GENBANK/AF277305
  • GENBANK/AF277306
  • GENBANK/AF277307
  • GENBANK/AF277308
  • GENBANK/AF277309
  • GENBANK/AF277310
  • GENBANK/AF277311
  • GENBANK/AF277312
  • GENBANK/AF277313
  • GENBANK/AF277314