title: Development and validation of a real-time PCR assay for a novel HTLV-1 tax sequence detection and proviral load quantitation.
abstract: A quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detect and quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers were designed, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral load was normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, the qPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assay reproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutions of the standard was 2.97 copies. The assay had an excellent dynamic range from 10⁵ to 10¹ copies per reaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performance of the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers ranged from 2.2×10² to more than 8.3×10⁴ copies/10⁶ PBMCs. The high sensitivity and wide dynamic range allowed the determination of a broad range of HTLV-1 proviral loads in infected individuals. This assay is a valuable alternative diagnostic tool when current available serological assays are insufficient. In addition, it will facilitate the study of the relationship between proviral load and pathogenesis.
authors.affiliation: Laboratorio de Virus Linfotrópicos Humanos-Retrovirus HIV-HTLV, Instituto de Virología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Enfermera Gordillo s/n, Ciudad Universitaria, CP: X5000HUA Córdoba, Argentina. gmcastro_arg@hotmail.com
authors.forename: Gonzalo M
authors.initials: GM
authors.lastname: Castro
authors.forename: Marcos C
authors.initials: MC
authors.lastname: Balangero
authors.forename: Eduardo
authors.initials: E
authors.lastname: Maturano
authors.forename: Arnaldo
authors.initials: A
authors.lastname: Mangeaud
authors.forename: Sandra V
authors.initials: SV
authors.lastname: Gallego
mesh_terms: D017931:DNA Primers / Q000235:genetics; D016355:Genes, pX; D015490:HTLV-I Infections / Q000175:diagnosis* / Q000821:virology*; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods* / Q000592:standards
chemical_list: D017931:DNA Primers