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 50 microliters 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, 7.5 U of
reverse transcriptase, and IU of thermostable
Taq DNA polymerase. The
reagent mixture was incubated for 15 min at 37 degrees C for RT followed by a variable amount of cycles of two-step PCR amplification (92 degrees C for 60 sec, 53 degrees C 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 10(2.8) TCID 50/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.