Figure 1
RT-PCR analysis of DENV-1, -2, -3 and 4 infected HPMEC-ST1.6R cells. Cell culture media of HPMEC-ST1.6R cells infected with DENV-1, -2, -3 and -4 were collected and used to obtain total RNA. A real-time RT-PCR analysis of HPMECST1.6R cells infected with DENV-1, -2, -3 and -4 were subsequently performed using the extracted RNA for quantitative analysis, using the MXPro3000P [Stratagene]. A SYBR Green 1 kit [Stratagene] and the DN-F/DN-R primer set [10] were utilized for amplifications, following instructions specified by the manufacturer. Following amplification, melting curve analysis was performed by raising the incubation temperature from 62°C to 95°C to verify correct amplification product by its specific melting temperature. DENV specific products derived from Vero or HPMEC cells infected with DENV-1 displayed a Tm of 80.7°C, while DENV-2, -3 and -4 displayed specific products with Tm values of 81.8°C, 80.1°C and 81.2°C, respectively.