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Amplificación de la región 5´UTR-C del genoma de los cuatro serotipos de Virus Dengue

Author(s): Daria Elena Camacho | Elizabeth Ferrer | Juana Ledia Triana-Alonso | Ana Celia Ferreras | Héctor Graterol | Guillermo Comach | Francisco Triana-Alonso

Journal: Salus Online
ISSN 1316-7138

Volume: 16;
Issue: 3;
Start page: 61;
Date: 2012;
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Keywords: dengue virus | dengue | RT-PCR | 5`UTR

Dengue virus infections (DENV) are the most important viral disease in terms of morbidity and mortality. The DENV genome is a single-stranded RNA with two untranslated regions (UTR) at the ends (5' UTR and 3' UTR) flanking an open reading frame (ORF). The 5 UTR and 3' UTR are important in the mechanisms of viral replication and protein synthesis, making them potential targets for regulation or inhibition of such processes by antiviral molecules. The objective of this work was to amplify the 5'UTR-C (Untranslated Region-Capsid) of the genome of the four DENV serotypes after optimizing the Reverse Transcription coupled to Polymerase Chain Reaction (RT-PCR), which could be used to assess viral translation processes in eukaryotic systems in vitro and its inhibition by potential antiviral molecules. Strains of different serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) were used and assays were performed with different concentrations of the primers and Taq polymerase and reverse transcriptase enzymes. The results indicated that the best response was obtained with primer concentrations of 0.5 µmol/L (DENV-1 and DENV-3), 1 µmol/L (DENV-2) and 0.75 µmol/L (DENV-4). The enzymes used showed high efficiency at the lowest quantity tested (1.25 U). According to the specified conditions, products were obtained from the 5'UTR-C with RT-PCR robust and reliable for amplification of these products
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