Abstract

USME-CIRO, José A.; GOMEZ-CASTANEDA, Alba M.  and  GALLEGO-GOMEZ, Juan C.. Molecular detection and typing of dengue virus by RT-PCR and nested PCR using degenerated oligonucleotides. Salud, Barranquilla [online]. 2012, vol.28, n.1, pp.1-15. ISSN 0120-5552.

Objective: To modify the commonly used primers for detecting and typing of dengue viruses and evaluate their performance on RNAs derived from clinical samples. Materials and methods: To determine the genetic variability within each dengue virus serotype, sequences of C-prM/M region available on GenBank were aligned allowing modifications to the primers conventionally used for virus typing. Modifications include nucleotide insertion and deletion in the 3' end taking into account the codon position, and also the inclusion of degenerated sites in highly variable positions. Primers were evaluated at different concentrations on extracted RNAs from infected cell cultures and subsequently from sera of probable dengue diagnosed patients. Results: All primers specifically amplified each dengue virus serotype from cell culture supernatants and clinical samples in a pilot test. Conclusions: The re-designed primers took into account the accumulated variability during more than 15 years, experimentally showing their value and utility for detecting and typing of dengue viruses.

Keywords : RT-PCR; nested PCR; degenerated primers; typing; dengue virus; molecular detection.

        · abstract in Spanish     · text in Spanish     · Spanish ( pdf )