Portal de Periódicos da CAPES

Microplate-reverse hybridization method to determine dengue virus serotype

1998; Elsevier BV; Volume: 73; Issue: 2 Linguagem: Inglês

10.1016/s0166-0934(98)00040-8

1879-0984

Tjahjani Mirawati Sudiro, Hiroaki Ishiko, Alan L. Rothman, Diana E Kershaw, Sharone Green, David W. Vaughn, Ananda Nisalak, Siripen Kalayanarooj, Francis A. Ennis,

Malaria Research and Control

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.