Quantitative detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells by SYBR green real-time PCR technique

J Clin Virol. 2004 Apr;29(4):282-9. doi: 10.1016/S1386-6532(03)00169-0.

Abstract

Background: The persistence of proviral human immunodeficiency virus type 1 (HIV-1) DNA reservoir represents one of the major drawbacks to the total eradication of HIV-1. The quantitative determination of proviral HIV-1 DNA load offers significant therapeutic information, especially when the HIV-1 RNA levels drop below the detectable limits during the highly active retroviral therapy (HAART) treatment. Moreover, the detection of HIV-1 proviral DNA is an important diagnostic marker in the evaluation of HIV-1 infection of newborns of HIV-1 seropositive women.

Objective: We evaluated a real-time PCR based on LightCycler technology revealed through SYBR green fluorochrome (SYBR green real-time PCR) to quantify the HIV-1 proviral DNA load in peripheral blood mononuclear cells (PBMC) of HIV-1 seropositive patients.

Study design: Firstly, we assessed the SYBR green real-time quantitative PCR for HIV-1 proviral DNA load detection determining the specificity and sensitivity of the assay using the LightCycler system. Secondly, we tested the performance of our SYBR green real-time PCR on 50 HIV-1 seropositive patients under HAART and 20 blood donors.

Results/conclusions: The results of this study showed that our SYBR green real-time PCR is able to detect five copies of the HIV-1 genome. Moreover, our method revealed HIV-1 proviral DNA in all the 50 HIV-1 seropositive patients ( 627 +/- 1068 HIV-1 proviral DNA copies per 10(6) PBMC, with a range of 30-6300 copies), whereas no positive signal was observed in any PBMC blood donors. Our SYBR green real-time PCR represents a sensitive and useful approach that could be applied both in HIV-1 proviral DNA reservoir determination and in HAART monitoring, particularly when the HIV-1 plasmatic RNA is undetectable.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Donors
  • CD4 Lymphocyte Count
  • DNA, Viral / analysis
  • DNA, Viral / isolation & purification*
  • HIV Infections / virology*
  • HIV Seropositivity / virology
  • HIV-1 / genetics
  • HIV-1 / isolation & purification*
  • Humans
  • Leukocytes, Mononuclear / virology*
  • Organic Chemicals*
  • Polymerase Chain Reaction / methods*
  • Proviruses / isolation & purification*
  • RNA, Viral / blood
  • Sensitivity and Specificity
  • Viral Load
  • Viremia

Substances

  • DNA, Viral
  • Organic Chemicals
  • RNA, Viral
  • SYBR Green I