{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2022,3,31]],"date-time":"2022-03-31T10:54:23Z","timestamp":1648724063069},"reference-count":25,"publisher":"FapUNIFESP (SciELO)","issue":"2","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Rev. Inst. Med. trop. S. Paulo"],"published-print":{"date-parts":[[1997,3]]},"abstract":"<jats:p>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 \u00b5l 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 37\u00baC for reverse transcription followed by 30 cycles of two-step PCR amplification (92\u00baC for 60 seconds, 53\u00baC 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<\/jats:p>","DOI":"10.1590\/s0036-46651997000200003","type":"journal-article","created":{"date-parts":[[2005,5,9]],"date-time":"2005-05-09T18:13:53Z","timestamp":1115662433000},"page":"79-84","source":"Crossref","is-referenced-by-count":0,"title":["Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method"],"prefix":"10.1590","volume":"39","author":[{"given":"Luiz Tadeu Moraes","family":"FIGUEIREDO","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weber Chelli","family":"BATISTA","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Akira","family":"IGARASHI","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"530","reference":[{"key":"ref1","series-title":"ed. 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