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 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.
Sudiro
T M
TM
Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester 01655, USA.
Ishiko
H
H
Rothman
A L
AL
Kershaw
D E
DE
Green
S
S
Vaughn
D W
DW
Nisalak
A
A
Kalayanarooj
S
S
Ennis
F A
FA
D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000145:classification* / Q000302:isolation & purification*; D004996:Ethidium; D006801:Humans; D012367:RNA, Viral / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012703:Serotyping; D013194:Staining and Labeling
D012367:RNA, Viral; D004996:Ethidium