We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

Apresentamos neste trabalho uma técnica simplificada de RT-PCR para identificação de vírus de dengue. Cinco estirpes brasileiras de vírus de dengue dos tipos 1 e 2, isoladas de pacientes e o vírus da vacina de febre amarela como controle negativo, foram utilizadas nos testes. Células C6/36 foram infectadas e os fluidos de cultura coletados após 7 dias. A RT-PCR, era efetuada em um único tubo contendo 1µl de fluidos infectados e diluídos 1/10 em água destilada ou em mistura detergente contendo Nonidet P40. A mistura de reação, num volume de 50 µl, continha 50 pmol de primers especificos, que amplificavam seqüência com 482 pares de bases para vírus do dengue tipo 1 e 210 pares de bases para dengue tipo 2, ou, ainda, primers para grupo dengue que amplificavam seqüência com 511 pares de bases. A mistura de reação, também, continha 0.1 mM dos 4 desoxinucleotídeos, 7.5 U de transcriptase reversa e 1 U de Taq DNA polimerase termoestável. A mistura era incubada por 5 minutos a 37º C para a transcrição reversa e processada em 30 ciclos de um PCR com 2 passos (92º C por 60 segundos, 53º C por 60 segundos) e incremento lento da temperatura. Produtos do PCR foram visualizados à luz ultra-violeta após eletroforese em gel de agarose e coloração em brometo de etídio. Baixos títulos virais como 103,6TCID50/ml foram detectados com o RT-PCR para dengue tipo 1. Amplificação genômica tipo-específica e grupo-específica para dengue foi obtida com todos os vírus do dengue estudados. Este RT-PCR, comparado a testes similares, é menos trabalhoso, mais rápido e possui reduzido risco de contaminação.

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination