Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus.

  • María Odreman Macchioli Universidad de Los Andes-Venezuela
  • Silvana Vielma Universidad de Los Andes- Venezuela
  • Daniel Atchley College of Pharmacy Harding University-USA
  • Guillermo Comacho
  • Alvaro Ramirez Instituto Venezolano de Investigaciones Científicas-Venezuela
  • Saberio Pérez Universidad de Los Andes-Venezuela
  • Luis Téllez Universidad de Los Andes-Venezuela
  • Beatriz Quintero Universidad de Los Andes-Venezuela
  • Erick Hernández Universidad de Los Andes-Venezuela
  • Maritza Muñoz Universidad de Los Andes-venezuela
  • José Mendoza Universidad de Los Andes-Venezuela
Palabras clave: dengue virus, qPCR amplification, acute phase diagnosis, NS5 gene region

Resumen

Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3”™-noncoding region (3”™NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3”™NC regions were 56.7% and 97%, respectively. Amplification concordance values be- tween C-prM/NS5 and NS5/3”™NC regions showed a weak (K= 0.109; CI 95%) and a moderate (K= 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan® format was much more sensitive than the C-prM/SYBR Green® I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples.

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Publicado
2013-04-10
Cómo citar
Odreman Macchioli, M., Vielma, S., Atchley, D., Comacho, G., Ramirez, A., Pérez, S., Téllez, L., Quintero, B., Hernández, E., Muñoz, M., & Mendoza, J. (2013). Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus. Investigación Clínica, 54(1). Recuperado a partir de https://produccioncientificaluz.org/index.php/investigacion/article/view/28905
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