Chapter title |
Proviral Load Determination of HTLV-1 and HTLV-2 in Patients' Peripheral Blood Mononuclear Cells by Real-Time PCR.
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Chapter number | 25 |
Book title |
Human Retroviruses
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Published in |
Methods in molecular biology, January 2014
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DOI | 10.1007/978-1-62703-670-2_25 |
Pubmed ID | |
Book ISBNs |
978-1-62703-669-6, 978-1-62703-670-2
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Authors |
Claudio Casoli, Elisabetta Pilotti, Umberto Bertazzoni, Casoli, Claudio, Pilotti, Elisabetta, Bertazzoni, Umberto |
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 %). |
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