Colorimetric Detection of Dengue by Single Tube Reverse-Transcription-Loop-Mediated Isothermal Amplification

Lau, Yee-Ling; Lai, Meng-Yee; Teoh, Boon-Teong; Abd-Jamil, Juraina; Johari, Jefree; Sam, Sing-Sin; Tan, Kim-Kee; AbuBakar, Sazaly

By using the primer sets selected, a single-tube one-step RT-LAMP assay system was standardized for rapid detection of all four serotypes of dengue viruses. The LAMP reaction was performed using plasmid DNA as template to determine the optimal reaction conditions. The amplification was observed as a typical ladder of multiple bands on the agarose gel. The monitoring of RT-LAMP assay amplification was carried out by naked-eye inspection following addition of HNB dye and real-time turbidimeter. Positive reaction was showed by a color change from violet to sky blue (Fig 1) . Temperature of 65°C was found to be optimal and the time of positivity observed through naked-eye and real-time monitoring using Loopamp realtime turbidimeter was found to be 30 min for DENV 1, 2 and 3 serotypes and 45 min for DENV 4 serotype (Fig 2) . The positive color (sky blue) and turbidity was only observed with the reference virus, whereas none of the control viruses showed a color change. Sensitivity of RT-LAMP detection