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

Abstract

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 microliters 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 degrees C for reverse transcription followed by 30 cycles of two-step PCR amplification (92 degrees C for 60 seconds, 53 degrees C 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.

A simple one‐step real‐time RT‐PCR for diagnosis of dengue virus infection

“…Several reverse transcriptase polymerase chain reaction (RT-PCR) methods have been developed in the past decade for detecting DENV infection during the acute phase [Deubel et al, 1990[Deubel et al, , 1997Lanciotti et al, 1992;Figueiredo et al, 1997aFigueiredo et al, , 1997b. However, the conventional RT-PCR is a laborious technique, which has a high risk of cross-contamination.…”

Duplex Reverse Transcription-PCR Followed by Nested PCR Assays for Detection and Identification of Brazilian Alphaviruses and Flaviviruses

“…To overcome such problems, some reverse transcription-PCR (RT-PCR) assays using species-specific primers have been described for certain alphaviruses (3,19,20,25,28,33,38,39) and certain flaviviruses (7,8,9,12,13,16,21,23,27,35). Since there are no specific clinical symptoms for arbovirus infection and different arboviruses are present in the same area, a universal genus-specific diagnosis by RT-PCR would be a useful tool for differential diagnosis of these infections.…”

Investigação sorológica de anticorpos IgM e IgG anti-dengue em crianças atendidas no Centro de Saúde Escola Dr. Edgard Aché do município de Ribeirão Preto,São Paulo

“…A confirmação laboratorial da infecção por DENV pode ser realizada através da detecção do vírus específico, do antígeno viral, seqüência genômica e/ou anticorpos feitas através de diversas técnicas sendo o isolamento viral, técnicas de biologia molecular e testes sorológicos os mais utilizados (LIMA et al, 2010;PUTTIKHUNT et al, 2011;HUANG, 2004 IGARASHI, 1997aIGARASHI, , 1997bLANCIOTTI et al, 1992).…”