title: A Luminex-based single DNA fragment amplification assay as a practical tool for detecting and serotyping dengue virus.
abstract: Dengue is a mosquito-borne viral infection that can evolve from subclinical to severe forms of disease. Early recognition during initial primary and secondary infections correlates with a reduced case-fatality rate in susceptible groups. The aim of this study was to standardize a DNA hybridization assay based on the Luminex technology for detecting and serotyping dengue virus (DENV). Reference DENVs representing the four different serotypes were used as controls to standardize the test. For validation, 16 DENV isolates obtained from a reference laboratory were analyzed in a double-blind manner to validate the test. Sixty blood samples from patients suspected of having dengue fever were used to evaluate the methodology after the validation step, and the results were compared with the reference semi-nested RT-PCR. Additionally, five human samples of each Zika and Chikungunya confirmed patients were used for specificity analysis. The Luminex-based assay correctly identified all 16 DENV isolates. In the evaluation step, the results of the RT-PCR/Luminex assay showed a concordance of 86.7% with those of the semi-nested RT-PCR. None of other virus infection samples was amplified. This is the first description of a hybridization assay that can discriminate the four DENV serotypes using probes against a single DENV sequence. The results indicated that the RT-PCR/Luminex DENV assay designed and evaluated in this study is a valuable additional tool for the early and rapid detection and serotyping of DENV, which could, in the future, be applied to new targets such as the Zika and Chikungunya viruses.
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Mauro Jorge
authors.initials: MJ
authors.lastname: Cabral-Castro
authors.affiliation: Departamento de Patologia, Faculdade de Medicina, Universidade Federal Fluminense, Niterói, RJ, Brazil.
authors.forename: Regina Helena Saramago
authors.initials: RHS
authors.lastname: Peralta
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Departamento de Doenças Infecciosas e Parasitárias, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Marta Guimarães
authors.initials: MG
authors.lastname: Cavalcanti
authors.affiliation: Laboratório de Líquido Cefalorraqueano, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Marzia
authors.initials: M
authors.lastname: Puccioni-Sohler
authors.affiliation: Instituto Evandro Chagas, Seção de Arbovirologia e Febres Hemorrágicas, Ananindeua, Pará, Brazil.
authors.forename: Valéria Lima
authors.initials: VL
authors.lastname: Carvalho
authors.affiliation: Instituto Evandro Chagas, Seção de Arbovirologia e Febres Hemorrágicas, Ananindeua, Pará, Brazil.
authors.forename: Pedro Fernando
authors.initials: PF
authors.lastname: da Costa Vasconcelos
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Electronic address: peralta@micro.ufrj.br.
authors.forename: José Mauro
authors.initials: JM
authors.lastname: Peralta
mesh_terms: D003715:Dengue / Q000175:diagnosis*; D003716:Dengue Virus / Q000145:classification* / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000379:methods*
keywords: Dengue virus; Early diagnosis; Molecular diagnosis; Polymerase chain reaction; RT-PCR/Luminex assay