A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load

Abstract

Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1-infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4(+) T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.

Latent human cytomegalovirus enhances HIV-1 infection in CD34+ progenitor cells

“…35 The instrument QuantStudio 3D Digital PCR system (Life Technologies) was used. Briefly, PCR reactions with templates were partitioned into chips with 20 000 wells and positive signals were assessed by a scanner.…”

Micro- and Nanotechnology for HIV/AIDS Diagnostics in Resource-Limited Settings

“…Many have also acknowledged that the proviral load may be an opportunity for early detection in adults in the 2-8 week window period [67] before seroconversion [61]- [63], a useful alternative for monitoring of ART when plasma RNA levels are undetectable [61], [68], [69], or a complement to the plasma RNA load for diagnostic or prognostic purposes [61], [68], [70]- [73]. Laboratory techniques for detection of proviral DNA have been described including nonquantitative PCR approaches [64], [74], quantitative PCR approaches [14], [61], [68], [69], [75], [76], electrochemiluminescencebased detection of PCR products [62], enzyme immunoassay detection of PCR products [63], and one study which performed PCR analysis on dried blood spots [60].…”

Joint production of prime/boost pairs of Fowlpox Virus and Modified Vaccinia Ankara recombinants carrying the same transgene

“…Two hours later the cells were washed and the complete medium restored. At various time points (18, 24, 48 and 64 h) the monolayer was washed and incubated with a lysis reagent described previously (Malnati et al, 2008) and proteinase K for 2 h at 56 °C, followed by a 15 min incubation at 95 °C. A fraction of the lysate was used in TaqMan Realtime PCR for FPV DNA quantitation.…”