title: Validation of a quantitative real-time PCR assay for HTLV-1 proviral load in peripheral blood mononuclear cells.
abstract: The objective of this study was to validate a TaqMan real-time PCR assay for HTLV-1 proviral load detection in peripheral blood mononuclear cells. TARL-2 cells were used to generate a standard curve. Peripheral blood mononuclear cell gDNA from 27 seropositive and 23 seronegative samples was analyzed. The sensitivity, specificity, accuracy, precision, dynamic range of the standard curve and qPCR efficiency were evaluated. All of the positive samples amplified the target gene. All of the negative samples amplified only the control gene (β-actin). The assay presented 100% specificity and sensibility. The intra- and inter-assay variability was 2.4% and 2.2%, respectively. The qPCR efficiency, slope and correlation coefficients (r2) were all acceptable. The limit of detection was 1 copy/rxn. This assay can reliably quantify HTLV-1 proviral load.
authors.affiliation: Laboratório de Líquido Cefalorraquiano, Serviço de Patologia Clínica, Hospital Universitário Clementino Fraga Filho (HUCFF)/Programa de Pós Graduação em Doenças Infecciosas e Parasitárias, Faculdade de Medicina,Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil. Electronic address: carolrosadas@gmail.com.
authors.forename: Carolina
authors.initials: C
authors.lastname: Rosadas
authors.forename: Mauro Jorge
authors.initials: MJ
authors.lastname: Cabral-Castro
authors.forename: Ana Carolina Paulo
authors.initials: AC
authors.lastname: Vicente
authors.forename: José Mauro
authors.initials: JM
authors.lastname: Peralta
authors.forename: Marzia
authors.initials: M
authors.lastname: Puccioni-Sohler
mesh_terms: D015490:HTLV-I Infections / Q000821:virology; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods
keywords: HTLV-1; PBMCs; Proviral load; Real-time PCR