1. Microplate-reverse hybridization method to determine dengue virus serotype.
- Author
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Sudiro TM, Ishiko H, Rothman AL, Kershaw DE, Green S, Vaughn DW, Nisalak A, Kalayanarooj S, and Ennis FA
- Subjects
- Dengue virology, Ethidium, Humans, RNA, Viral blood, Sensitivity and Specificity, Serotyping, Staining and Labeling, Dengue Virus classification, Dengue Virus isolation & purification, Reverse Transcriptase Polymerase Chain Reaction
- 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.
- Published
- 1998
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