Microplate-reverse hybridization method to determine dengue virus serotype

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 R...

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Bibliographic Details
Published in:Journal of virological methods 1998-08, Vol.73 (2), p.229-235
Main Authors: Sudiro, T.Mirawati, Ishiko, Hiroaki, Rothman, Alan L, Kershaw, Diana E, Green, Sharone, Vaughn, David W, Nisalak, Ananda, Kalayanarooj, Siripen, Ennis, Francis A
Format: Article
Language:English
Subjects:
Biological and medical sciences
Dengue - virology
Dengue virus
Dengue Virus - classification
Dengue Virus - isolation & purification
Ethidium
Fundamental and applied biological sciences. Psychology
Humans
Microbiology
Microplate hybridization
Reverse Transcriptase Polymerase Chain Reaction
RNA, Viral - blood
RT-PCR
Sensitivity and Specificity
Serotyping
Staining and Labeling
Techniques used in virology
Virology
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Summary: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.
ISSN:0166-0934
1879-0984
DOI:10.1016/S0166-0934(98)00040-8