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 %).
An erratum to this chapter is available at http://dx.doi.org/10.1007/978-1-62703-670-2_27
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-62703-670-2_27
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Acknowledgments
This work was supported in part by AIRC 2008-Cariverona Regional grant (to U. Bertazzoni) and in part by Istituto Superiore di Sanità, National Research Project on AIDS (grants 40D.14 and 40D.62, ELVIS Italian Network on LTNP, 2007), and EC Project number LSHP-CT-2007-037616 (Genetic and Immunological Studies of European and African HIV-1+ Long Term Non-Progressors, GISHEAL) (to C. Casoli).
The authors express their thanks to Marco Turci and Cecilia Bender for useful information on proviral load determination.
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Casoli, C., Pilotti, E., Bertazzoni, U. (2014). Proviral Load Determination of HTLV-1 and HTLV-2 in Patients’ Peripheral Blood Mononuclear Cells by Real-Time PCR. In: Vicenzi, E., Poli, G. (eds) Human Retroviruses. Methods in Molecular Biology, vol 1087. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-670-2_25
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DOI: https://doi.org/10.1007/978-1-62703-670-2_25
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