Development and validation of a real-time PCR assay for a novel HTLV-1 tax sequence detection and proviral load quantitation

ELSEVIER SCIENCE BV

Año: 2013 p. 383 - 383

quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detectand quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers weredesigned, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral loadwas normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, theqPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assayreproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutionsof the standard was 2.97 copies. The assay had an excellent dynamic range from 105 to 101 copies perreaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performanceof the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers rangedfrom 2.2×102 to more than 8.3