Microplate-reverse hybridization method to determine dengue virus serotype

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

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

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. Copyright (C) 1998 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)229-235
Number of pages7
JournalJournal of Virological Methods
Volume73
Issue number2
DOIs
Publication statusPublished - 1 Aug 1998

Keywords

  • Dengue virus
  • Microplate hybridization
  • RT-PCR

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