Quantification of Human Immunodeficiency Virus Type 1 Proviral DNA by Using TaqMan Technology

Abstract

A protocol for quantification of human immunodeficiency virus type 1 (HIV-1) proviral DNA with the TaqMan technology was developed and validated. The assay was specific for HIV-1, with an analytic sensitivity of 10 copies and a linear dynamic range of >6 logs. Viral RNA levels, when at a stable state, were highly correlated with proviral DNA levels in 80 specimens of 18 HIV-infected children.The use of highly active antiretroviral therapy (HAART) in human immunodeficiency virus (HIV)-infected patients can often significantly reduce the levels of viral RNA to undetectable levels in plasma in both adults and children (13)(14)(15)(16)(17). However, it is less clear how changes in proviral DNA levels respond to HAART in infected patients or what relative changes in proviral DNA levels precede changes in viral RNA levels after HAART induction. One of the current challenges is detection of low levels of proviral DNA in latently infected CD4 ϩ lymphocytes and other reservoirs, which can replenish and revive viral infection upon activation (3-5). Thus, a highly reproducible and accurate assay for quantification of proviral DNA would enable more in-depth evaluations of the efficacies of antiviral therapies.Several assays for the quantification of HIV type 1 (HIV-1) proviral DNA have previously been reported, and all of these were based on the principle of conventional PCR (1, 2, 6, 10, 11). The potential limitation associated with the traditional quantitative PCR is that the DNA copy numbers are calculated on the basis of the quantities of the final amplified gene products. Since DNA is amplified exponentially during PCR, a small variation in amplification efficiency early in the thermocycling process could potentially lead to large variations in the final quantities of amplified products.The real-time PCR, which is also known as the TaqMan or the 5Ј exonuclease assay, quantifies PCR products cycle by cycle (in real time) as they accumulate (7-9). There are several advantages of using real-time PCR. (i) It does not rely on the final product of PCR amplification for quantification. DNA copy numbers are determined on the basis of the threshold cycle number (C T ), which is directly proportional to the initial copy number. (ii) It is relatively specific. If the probe binds nonspecifically to some sequences other than the target sequence, it will not be cleaved or detected as part of the amplification. (iii) It allows a wide dynamic range of detection since the measured DNA copy number is directly proportional to the initial copy number. (iv) All real-time PCR tests are performed in 96-well and closed-tube formats, allowing highthroughput testing and a greatly reduced chance of cross-contamination.In this report, a new protocol for quantification of HIV-1 proviral DNA with the TaqMan technology is described and validated. By using this method, HIV-1 proviral DNA levels were compared in parallel with the HIV-1 RNA levels at a stable state during HAART.(This study was part of a thesis by Johann Miller in partial fulfillment of...

Histone Deacetylase 6 Regulates Human Immunodeficiency Virus Type 1 Infection

“…When indicated, T-cells were pretreated with anti-CD4 mAbs (5 g/ml), SDF-1␣ (300 nM), 3Ј-azido-2Ј,3Ј-dideoxythymidine (AZT) (5 g/ml), TSA (dose response), or NaBut (500 M). HIV-1 entry into parental or transfected MT-2 cells was monitored 12 h after HIV-1 infection by measuring proviral gag HIV-1 DNA by PCR (Zhao et al, 2002). HIV-1 infection (MOI, 0.1) of PHA-activated PBLs was assessed every 48 h by measuring the concentration of p24 in the culture supernatant by enzyme-linked immunosorbent assay (INNOTEST HIV-1 antigen mAb; Innogenetic, Ghent, Belgium).…”

Transfer and Evaluation of an Automated, Low-Cost Real-Time Reverse Transcription-PCR Test for Diagnosis and Monitoring of Human Immunodeficiency Virus Type 1 Infection in a West African Resource-Limited Setting

“…In developed countries, these assays have been mainly evaluated for viruses that are not of high commercial interest, such as human herpesvirus 8 (4), human T-cell leukemia type 1 (8), cytomegalovirus (23), and adenovirus (22), as well as for hepatitis B virus (17), and hepatitis C virus (41). For HIV infection, the assays have been used for HIV-2 (7), HIV-1 group O (16), and HIV-1 DNA proviral load (10,12,44). Moreover, plasma HIV-1 RNA has been assessed by real-time PCR for multiplex nucleic acid amplification testing in blood donations (25,26,40) and for HAART monitoring in HIV-1-seropositive patients (15,30).…”

Update on the laboratory diagnosis and monitoring of HIV infection

“…Since DNA is amplified exponentially during PCR, a small variation in amplification efficiency early in the thermocycling process could potentially lead to a large variation in the final quantification of amplified products. To circumvent this problem, a TaqMan-based protocol for quantification of HIV-1 proviral DNA was reported [24]. This assay is based on the principle of real-time PCR, which is also known as TaqMan TM [25].…”

“…This assay is based on the principle of real-time PCR, which is also known as TaqMan TM [25]. Due to the inherent advantage of real-time PCR over classic PCR, this new assay provides a highly accurate and reproducible detection method for HIV-1 proviral DNA with a broad linear range of detection [24].…”