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. 2015 Sep 18;10(9):e0138694.
doi: 10.1371/journal.pone.0138694. eCollection 2015.

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

Yee-Ling Lau  1 , Meng-Yee Lai  1 , Boon-Teong Teoh  2 , Juraina Abd-Jamil  2 , Jefree Johari  2 , Sing-Sin Sam  2 , Kim-Kee Tan  2 , Sazaly AbuBakar  2
Affiliations

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

Yee-Ling Lau et al. PLoS One. .

Abstract

Dengue is usually diagnosed by isolation of the virus, serology or molecular diagnostic methods. Several commercial kits for the diagnosis of dengue are existing, but concerns have arisen regarding to the affordability and performance characteristics of these kits. Hence, the loop-mediated isothermal amplification (LAMP) is potentially ideal to be used especially in resource limited environments. Serum was collected from healthy donors and patients diagnosed with dengue infection. RNA extracted from the serum samples were tested by reverse-transcription-LAMP assay developed based on 3'-NCR gene sequences for DENV 1-4. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. Sensitivity and specificity of RT-LAMP results were calculated and compared to qRT-PCR and ELISA. RT-LAMP is highly sensitive with the detection limit of 10 RNA copies for all serotypes. Dengue virus RNA was detected in all positive samples using RT-LAMP and none of the negative samples within 30-45 minutes. With continuing efforts in the optimization of this assay, RT-LAMP may provide a simple and reliable test for detecting DENV in areas where dengue is prevalent.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Visualization of LAMP assay products following addition of HNB dye.
The color changes from violet (negative reaction) to sky blue (positive reaction). Tube A and B: DENV 1 serotypes; tube C and D: DENV 2 serotypes; tube E and F: DENV 3 serotypes; tube G: DENV 4 serotype; tube H: JEV; tube I: CHIKV; tube J: SINV; tube K: Negative control (distilled water).
Fig 2
Fig 2. Optimal time of detection for DENV 1, DENV 2, DENV 3 and DENV 4 serotypes.
Panel A-C: optimal time of detection for DENV 1–3 serotypes is 30 min; panel D: optimal time of detection for DENV 4 serotype is 45 min.
See this image and copyright information in PMC

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