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Quantitation of HTLV-I and II proviral load using real-time quantitative PCR with SYBR Green chemistry.
- Author(s): Lee, Tzong-Hae;
- Chafets, Daniel M;
- Busch, Michael P;
- Murphy, Edward L
- et al.
Published Web Locationhttp://www.journalofclinicalvirology.com/article/S1386-6532(04)00167-2/abstract
No data is associated with this publication.
BackgroundHuman T-lymphotropic virus type I (HTLV-I) is linked etiologically with adult T cell leukemia/lymphoma and HTLV-I-associated myelopathy/tropical spastic paraparsis (HAM/TSP). Human T-lymphotropic virus type II (HTLV-II) is associated with HAM/TSP and, in HIV coinfected patients only, rare cases of cutaneous T cell lymphoma. Proviral load may be important in the pathogenesis of HTLV-associated disease.
Materials and methodsA real time quantitative PCR assay using SYBR Green intercalation was established. Primers targeting the tax region were standardized against MT2 and MOT cell line DNA for HTLV-I and HTLV-II, respectively. HTLV-I/II copy number was normalized to the amount of cellular DNA by quantitation of the HLA-DQ alpha gene. We measured proviral load in peripheral blood mononuclear cells (PBMCs) in a large cohort of 120 HTLV-I and 335 HTLV-II seropositive former blood donors. We also assessed the intra- and inter-assay reproducibility of the assay.
ResultsProviral load for HTLV-I infected patients ranged from 3.1 x 10(0) to 1.8 x 10(5)copies/10(6) PBMCs with a mean of 1.6 x 10(4) and a median of 3.0 x 10(3). HTLV-I was undetectable in 7 of 120 cases (5.8%). Proviral load for HTLV-II infected patients ranged from 1.1 x 10(0) to 1.0 x 10(6)copies/10(6) PBMCs with a mean of 2.8 x 10(4) and a median of 5.0 x 10(2). HTLV-II was undetectable in 31 out of 335 cases (9.3%).
ConclusionThe assay has excellent dynamic range from 10(6) to 10(0)copies/reaction, good intra- and inter-assay reproducibility, and a lower limit of detection of a single copy per reaction. The sensitivity and high dynamic range allow determination of a broad range of HTLV-I/II proviral load in clinical subjects. This assay will facilitate the study of the relationship between proviral load and pathogenesis.
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