Validation of a quantitative real-time PCR assay for HTLV-1 proviral load in peripheral blood mononuclear cells |
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| Authors: | Carolina Rosadas,Mauro Jorge Cabral-Castro,Ana Carolina Paulo Vicente,José Mauro Peralta,Marzia Puccioni-Sohler |
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| Affiliation: | 1. 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;2. Laboratório de Diagnóstico Imunológico e Molecular de Doenças Infecciosas e Parasitárias/Programa de Pós Graduação do Instituto de Microbiologia Paulo Góes, UFRJ, Rio de Janeiro, Brazil;3. Laboratório de Genética de Microorganismos, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil;4. Escola de Medicina e Cirurgia, Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Brazil |
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| 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. |
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| Keywords: | HTLV-1 Real-time PCR Proviral load PBMCs |
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