Identification of Brazilian flaviviruses by a simplified reverse transcription polymerase chain reaction method using Flavivirus universal primers

被引:19
作者
Figueiredo, LTM
Batista, WC
Kashima, S
Nassar, ED
机构
[1] Univ Sao Paulo, Fac Med Ribeirao Preto, Unidade Multidisciplinar Pesquisa Virol, BR-14049900 Ribeirao Preto, SP, Brazil
[2] Fundacao Hemoctr Ribeirao Preto, BR-14051140 Ribeirao Preto, SP, Brazil
[3] Adolfo Lutz Inst, Serv Virol, BR-01246902 Sao Paulo, Brazil
关键词
D O I
10.4269/ajtmh.1998.59.357
中图分类号
R1 [预防医学、卫生学];
学科分类号
1004 ; 120402 ;
摘要
We report a simplified reverse transcription-polymerase chain reaction (RT-PCR) method for identification of Brazilian flaviviruses based on the patterns of electrophoretic separation of the amplicons. The RT-PCR was done on the culture fluids of Aedes albopictus C6/36 cells infected with Brazilian flaviviruses, without previous extraction of viral RNA, using Flavivirus universal primers that anneal to highly conserved sequences within the nonstructural protein 5 and 3'- non translated region of the virus genome. Genomes of 13 Brazilian Flavivirus isolates were amplified. It was not possible to amplify the genome of Bussuquara virus. Analysis of the RT-PCR products gave reproducible results and three distinct amplicon patterns were observed. Cacipacore (800-850 basepairs [bp]) and yellow fever viruses (600 bp) yielded a single amplicon; dengue virus types 1 and 2 (650 and 550 bp), dengue virus type 4 (550 and 450 bp), Iguape (650-600 bp and 750-700 bp), St. Louis encephalitis (700 and 650-600 bp), and Rocio viruses (600 and 500-550 bp) yielded two amplicons; and Ilheus virus yielded five amplicons, two larger than 1,000 bp, one 650-700 bp, one 550-600 bp, and one 450-500 bp. The analysis of amplicon DNA sequences of six viruses showed homology with the 3'- nontranslated region of Flavivirus genome. The use of the Flavivirus universal primers in this simple RT-PCR technique is suitable as a screening test for the genus Flavivirus, with the exception of Bussuquara virus, in Brazilian isolates in tissue culture fluid.
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页码:357 / 362
页数:6
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