1. Monitoring of HTLV-1-associated diseases by proviral load quantification using multiplex real-time PCR
- Author
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Evandra Strazza Rodrigues, Suellen Salustiano, Elaine Vieira Santos, Svetoslav Nanev Slavov, Virgínia Picanço-Castro, Juliana Matos Maçonetto, Tissiana Marques de Haes, Osvaldo Massaiti Takayanagui, Dimas Tadeu Covas, and Simone Kashima
- Subjects
Human T-lymphotropic virus 1 ,beta-Globins ,Viral Load ,Real-Time Polymerase Chain Reaction ,HTLV-I Infections ,Paraparesis, Tropical Spastic ,Cellular and Molecular Neuroscience ,Proviruses ,Neurology ,Virology ,DNA, Viral ,Leukocytes, Mononuclear ,Humans ,Neurology (clinical) - Abstract
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
- Published
- 2022