Rapid discriminative detection of dengue viruses via loop mediated isothermal amplification

Abstract

Dengue virus (DENV) is one of the life-threatening viruses to the human. In this study, we have designed specific novel primers for rapid discriminative detection of DENV-1, DENV-2, and DENV-4 by real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction. The effect of parameters such as reaction temperature and magnesium sulfate was investigated on the RT-LAMP reaction for detection of DENV RNA. Under the optimal conditions, this method is able to differentiate and to detect DENV within 25 min, exhibiting detection limit of 3.5 copies/μL. Importantly, the novel specific primers-based RT-LAMP assay did not react with other viruses, suggesting the selectivity of the method towards DENV RNA. The RT-LAMP reaction products are easily visualized with naked-eye when irradiated them under UV light at 365 nm. Amplification products could be visualized directly for color changes. This method provides a facile, and accurate molecular amplication technique for the rapid discriminative detection of dengue viruses. The RT-LAMP platform can be used as a promissing diagnostic tool for discriminative detection of DENV without aid of complicated protocols or sophisticated equipment.