title: Proviral load determination of HTLV-1 and HTLV-2 in patients' peripheral blood mononuclear cells by real-time PCR.
abstract: TaqMan real-time PCR assays were developed to determine the proviral load (PVL) of human T-cell leukemia viruses type 1 and 2 (HTLV-1 and HTLV-2) in peripheral blood mononuclear cells (PBMCs) of infected subjects. In particular, separate single-plex assays for HTLV-1 tax-1, and HTLV-2 tax-2 and pol-2 genes were designed for quantitation of HTLV-1 and HTLV-2 PVLs. The specificity of both tax-2 and pol-2 assays was verified by testing the DNA extracted from C10, a chronically HTLV-1-infected cell line, used as a negative control. As far as HTLV-2 assay, the specificity was checked by testing C344 cells which are stably infected by HTLV-2. Quantitative determination of HTLV PVLs was obtained by performing standard reference curves by a serial dilution of DNA extracted from C10 and C344 cells, assuming one proviral genome per C10 cell and two per C344 cell. The human albumin gene, of which there are 2 copies per cell, was quantified in the same reactions to normalize the results. Intra-assay reproducibility was checked by running 30 replicates of the same sample in a plate (coefficient of variance <6 %), while inter-assay reproducibility was measured by amplifying the same sample in three independent experiments (coefficient of variance <6 %).
authors.affiliation: Department of Clinical Sciences, University of Milano, Milano, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Casoli
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Pilotti
authors.forename: Umberto
authors.initials: U
authors.lastname: Bertazzoni
mesh_terms: D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000502:physiology*; D015367:Human T-lymphotropic virus 2 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D011533:Proviruses / Q000235:genetics / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019562:Viral Load