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

The serotype-specific oligonucleotide primers used for RT-LAMP assay amplification of dengue viruses were designed using the Primer-Explorer V3 software based on 3 0 noncoding region (NCR) (GenBank accession number: DENV-1 Western Pacific, U88535; DENV-2 New Guinea C, AF038403; DENV-3 H87, M93130, and DEN-4 China Guangzhou B5, AF289029) ( Table 1 ) [6] . Loop primers (Loop-F and Loop-B) were designed manually. The RT-LAMP assay was performed using Loopamp RNA amplification kit (Eiken Chemical Co. Ltd., Japan). Briefly, a 25 μl reaction mixture consisted of 1.6 μM FIP and BIP primer, 0.8 μM Loop-F and Loop-B primer, 0.2 μM F3 and B3 primer, 12.5 μl of 2X reaction mixture (RM) (provided in the kit), 1 μl of Enzyme mixture (EM) (provided in the kit), 0.7 μl of sterile deionize water, 1μl of Florescent Detection reagent (FD) (provided in the kit) and 1 μl of template RNA. Single tube RT-LAMP was conducted by adding all primer sets in a single reaction mixture. The reaction mixture was carried out in a Loopamp real-time turbidimeter (LA-320, Teramecs, Co., Ltd., Japan). To prevent cross-contamination, different sets of pipettes and different work areas were designated for template preparation, reaction mixture preparation and DNA amplification. All experiments were performed in duplicate at least 2 times.