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


METHODS
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.


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.
Gibellini
Davide
D
Department of Clinical and Experimental Medicine, Microbiology Section, University of Bologna, St. Orsola Hospital, Via Massarenti 9, 40138 Bologna, Italy. davide.gibellini@unibo.it
Vitone
Francesca
F
Schiavone
Pasqua
P
Ponti
Cristina
C
La Placa
Michele
M
Re
Maria Carla
MC
D052160:Benzothiazoles; D001782:Blood Donors; D018791:CD4 Lymphocyte Count; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D003959:Diamines; D015658:HIV Infections / Q000821:virology*; D006679:HIV Seropositivity / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000302:isolation & purification*; D011804:Quinolines; D012367:RNA, Viral / Q000097:blood; D012680:Sensitivity and Specificity; D019562:Viral Load; D014766:Viremia
D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I