期刊
JOURNAL OF NEUROVIROLOGY
卷 28, 期 1, 页码 27-34出版社
SPRINGER
DOI: 10.1007/s13365-020-00924-2
关键词
HTLV-1; Proviral load
资金
- Financiadora de Estudos e Projetos (FINEP) [1387/10]
- Centro de Terapia Celular (CTC/FAPESP)/Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2013/08135-2]
- Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [465539/2014-9]
- Fundacao Hemocentro de Ribeirao Preto (FUNDHERP)
In this study, a sensitive multiplex real-time PCR method was successfully optimized for the simultaneous detection and quantification of HTLV-1 proviral load and endogenous gene. The method was found to be effective in monitoring and following up the clinical progression of HTLV-1 disease in infected individuals.
Proviral load (PVL) is one of the determining factors for the pathogenesis and clinical progression of the human T-lymphotropic virus type I (HTLV-1) infection. In the present study, we optimized a sensitive multiplex real-time PCR for the simultaneous detection and quantification of HTLV-1 proviral load and beta-globin gene as endogenous control. The values obtained for HTLV-1 PVL were used to monitor the clinical evolution in HTLV-1-infected individuals. A vector containing cloned DNA targets of the real-time PCR for the beta-globin gene and the HTLV-1pol region was constructed. For the reaction validation, we compared the amplification efficiency of the constructed vector and MT-2 cell line containing HTLV-1. The analytical sensitivity of the reaction was evaluated by the application of a standard curve with a high order of magnitude. PVL assay was evaluated on DNA samples of HTLV-1 seropositive individuals. The construct showed adequate amplification for the beta-globin and HTLV-1 pol genes when evaluated as multiplex real-time PCR (slope = 3.23/3.26, Y-intercept = 40.18/40.73, correlation coefficient r(2) = 0.99/0.99, and efficiency = 103.98/102.78, respectively). The quantification of PVL using the MT-2 cell line was equivalent to the data obtained using the plasmidial curve (2.5 copies per cell). In HTLV-1-associatedmyelopathy/tropical spastic paraparesis patients, PVL was significantly higher (21315 +/- 2154 copies/10(5) PBMC) compared to asymptomatic individuals (1253 +/- 691 copies/10(5) PBMC). The obtained results indicate that the optimized HTLV-1 PVL assay using plasmidial curve can be applied for monitoring and follow-up of the progression of HTLV-1 disease. The use of a unique reference plasmid for both HTLV-1 and endogenous gene allows a robust and effective quantification of HTLV-1 PVL. In addition, the developed multiplex real-time PCR assay was efficient to be used as a tool to monitor HTLV-1-infected individuals.
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