title: Detection and identification of dengue-1 virus in clinical samples by a nested-PCR followed by restriction enzyme digestion of amplicons.
abstract: Dengue viruses cause a disease with clinical findings ranging from asymptomatic infections to severe manifestations, characterised by haemorrhage and shock and known as dengue haemorrhagic fever/dengue shock syndrome. Since this fever and syndrome usually results from sequential infections by distinct dengue serotypes, rapid detection and identification of dengue viruses circulating in endemic areas are important to implement control measures, and ultimately to avoid secondary infections that could result in dengue haemorrhagic fever/dengue shock syndrome. A nested-PCR was developed followed by restriction enzyme (Kpn I) digestion of the amplicons to differentiate dengue-1 from dengue-2. Seventy-five IgM-containing samples collected from 2 to 17 days after the beginning of the symptoms were examined. These samples were submitted to nested-PCR amplification, the amplicons were digested with Kpn I, and the results compared to virus isolation in C6/36 cells and to results obtained by the standard PCR. Out of 75 tested samples, virus was isolated from 2 (2.6%), 17 (22.7%) were positive by the regular PCR protocol, and 58 (77.3%) were positive by nested-PCR. All of the amplicons digested by Kpn I identified dengue-1 virus as the infecting strain. These results indicate that the nested-PCR provided a high yield of dengue genome amplification even in the presence of IgM antibodies, and restriction enzyme digestion defined rapidly the circulating serotype. Therefore, the combination of these techniques may be useful to rapidly identify dengue viruses in countries where dengue-1 and dengue-2 circulates, and this approach can also be applied to the other two serotypes.
authors.affiliation: Department of Internal Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil.
authors.forename: Sérgio Oliveira
authors.initials: SO
authors.lastname: De Paula
authors.forename: Danielle Malta
authors.initials: DM
authors.lastname: Lima
authors.forename: Benedito Antônio Lopes
authors.initials: BA
authors.lastname: da Fonseca
mesh_terms: D003715:Dengue / Q000175:diagnosis* / Q000821:virology*; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014776:Virus Cultivation
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific; C029312:GGTACC-specific type II deoxyribonucleases