Abstract
BackgroundThe human immunodeficiency virus type 1 (HIV-1) proviral DNA persists in infected cells, even after prolonged successful HAART. In the present study, a relative quantification assay of HIV-1 proviral DNA by LightCycler® real-time PCR based on SYBR Green I detection was developed in comparison to the number of purified CD4+ cells as estimated by the quantification of the β-globin gene.MethodsThe ability of the designed gag primers to quantify HIV-1 Group M and the PCR efficiency were assessed on HIV-1 reference isolate subtypes A, B, C and D. The 8E5 cell line containing a single defective copy of HIV-1 proviral DNA was used as a standard for both the HIV-1 target gene and the β-globin reference gene. The assay was applied on thirty consecutive patient samples received for RNA viral load determinations and on retrospective samples from fifteen patients undergoing 2 years of structured treatment interruption (STI).ResultsThe lower limit of quantification was 50 HIV-1 DNA proviral copies per CD4+ cell sample. The dynamic range was from 50 to 106 HIV-1 DNA copies per CD4+ cell sample with intra- and inter-assay coefficients of variability ranging from 3.1% to 37.1%. The β-globin reference gene was quantified down to a limit of 1.5 pg of DNA/μl (approximately 5 cells) with intra- and interassay coefficients of variability ranging from 1.8% to 21%. DNA proviral load varies widely among HIV-1 infected patients. Proviral load and plasma viral load rebound were high in STI patients who took longer to achieve an undetectable plasma viral load under therapy. A statistically significant correlation was observed between DNA proviral load and RNA steady state viral load in STI patients (p-value = 0,012).ConclusionWe have developed a fast, sensitive and specific relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells. The assay enables the monitoring of HIV-1 proviral load, which may be useful to monitor therapy efficacy especially in patients with undetectable plasma RNA viral load, and allows the exploration of viral reservoirs.
Highlights
The human immunodeficiency virus type 1 (HIV-1) proviral DNA persists in infected cells, even after prolonged successful HAART
The HIV-1 proviral DNA load could be an alternative viral marker, as it is known that proviral DNA persists in infected cells, even after prolonged successful HAART as evidenced by undetectable plasma RNA viral load
As 95 to 99% of infected cells are CD4+ cells[25], we developed a relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells on the LightCycler® by real-time PCR compared to cell quantification by β-globin PCR
Summary
The human immunodeficiency virus type 1 (HIV-1) proviral DNA persists in infected cells, even after prolonged successful HAART. A relative quantification assay of HIV-1 proviral DNA by LightCycler® real-time PCR based on SYBR Green I detection was developed in comparison to the number of purified CD4+ cells as estimated by the quantification of the β-globin gene. We used the 8E5 cell line containing a single defective copy of HIV-1 proviral DNA as a standard for both the HIV-1 target gene and the β-globin reference gene in order to carry out a relative quantification. Another recent publication described a real-time PCR based on LightCycler® technology revealed through SYBR green fluorochrome to quantify the HIV-1 proviral DNA load[24]. As 95 to 99% of infected cells are CD4+ cells[25], we developed a relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells on the LightCycler® by real-time PCR compared to cell quantification by β-globin PCR
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