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.
Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester 01655, USA.
T M
TM
Sudiro
H
H
Ishiko
A L
AL
Rothman
D E
DE
Kershaw
S
S
Green
D W
DW
Vaughn
A
A
Nisalak
S
S
Kalayanarooj
F A
FA
Ennis
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