Microplate-reverse hybridization method to determine dengue virus serotype

被引:10
作者
Sudiro, TM
Ishiko, H
Rothman, AL
Kershaw, DE
Green, S
Vaughn, DW
Nisalak, A
Kalayanarooj, S
Ennis, FA
机构
[1] Univ Massachusetts, Sch Med, Ctr Infect Dis & Vaccine Res, Worcester, MA 01655 USA
[2] Univ Indonesia, Fac Med, Dept Microbiol, Jakarta, Indonesia
[3] USA, Armed Forces Res Inst Med Sci, Med Component, Dept Virol, Bangkok, Thailand
[4] Bangkok Childrens Hosp, Dept Infect Dis, Bangkok, Thailand
关键词
dengue virus; RT-PCR; microplate hybridization;
D O I
10.1016/S0166-0934(98)00040-8
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 1998 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:229 / 235
页数:7
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