TY - JOUR
T1 - Microplate-reverse hybridization method to determine dengue virus serotype
AU - Sudiro, Tjahjani Mirawati
AU - Ishiko, Hiroaki
AU - Rothman, Alan L.
AU - Kershaw, Diana E.
AU - Green, Sharone
AU - Vaughn, David W.
AU - Nisalak, Ananda
AU - Kalayanarooj, Siripen
AU - Ennis, Francis A.
PY - 1998/8
Y1 - 1998/8
N2 - 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.
AB - 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.
KW - Dengue virus
KW - Microplate hybridization
KW - RT-PCR
UR - http://www.scopus.com/inward/record.url?scp=0032145928&partnerID=8YFLogxK
U2 - 10.1016/S0166-0934(98)00040-8
DO - 10.1016/S0166-0934(98)00040-8
M3 - Article
C2 - 9766894
AN - SCOPUS:0032145928
SN - 0166-0934
VL - 73
SP - 229
EP - 235
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 2
ER -