Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription-polymerase chain reaction (RT-PCR) method.

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