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

Loop-mediated isothermal amplification (LAMP) represents a novel DNA amplification method developed by Notomi et al. (2000) that within 1 h (at 65°C) effectively provided diagnostic results [3] . LAMP is easy to use, convenient, and cost-effective, requiring minimal equipment such as water baths or heating blocks. Therefore, it has been widely applied as a diagnostic tool for several viral, bacterial, and parasitic diseases [13] [14] [15] . Parida et al. (2005) reported a one-step RT-LAMP for the detection of 4 dengue serotypes (in separate tubes) by targeting the 3'-NCR [4] . In 2011, Li et al. developed a single tube reaction system for the detection of DENV infection using RT-LAMP primers based on amplification of the C-prM gene; however, this gene is relatively less well conserved among all 4 DENV serotypes (inter-serotype) [16] . These 2 studies evaluated their RT-LAMP assays based on their ability to detect DENV infections using a small clinical sample size (<70). In 2013, Teoh et al. reported a single-tube reverse RT-LAMP assay targeting the 3' untranslated region (UTR) for the detection of all 4 DENV serotypes in 171 confirmed dengue samples. However the sensitivity of this RT-LAMP assay was only 92.5% compared to qRT-PCR [17] . In the present study, an one step RT-LAMP assay was designed to amplify the 3'NCR capable of diagnosing the 4 dengue serotypes. In addition to the real-time monitoring provided by the present LAMP test, this study was the first to apply HNB dye for the detection of amplified targets using the naked eye following 30-45 min amplification step at 65°C. The amplification time for our RT-LAMP assay (based on the 3'NCR) was significantly shorter compared to previously developed RT-LAMP assays targeting the 3'UTR [17] . RT-LAMP assay designed in this study was shown to be 100% sensitive and specific which is better as compared to qRT-PCR (Table 3 ). The date of illness onset ranged from day 1 to day 11 of illness. DENV viral loads detected on day 1 to day 9 ranged from 2.97 to 6.67 log10 DENV RNA copies/ml of serum. Viral loads of 2.93 log10 RNA copies/ml (equivalent to 10 RNA copies) were beyond the detection limit of qRT-PCR [18] . This is one of the possible reasons of false-negative qRT-PCR results. The simplicity and high efficiency of LAMP to rapidly amplify DNA under isothermal conditions suggested that LAMP could be a potential alternative for detecting dengue virus especially in field settings as compared to qRT-PCR which is time-consuming and require RT-PCR machine [19] [20] [21] . Another advantage of using the LAMP assay is the colorimetric detection of positive reactions that allows positive and negative amplifications to be distinguished based on observed color changes with the naked eye.