A Multiplex RT-LAMP-Lateral Flow Platform For Comprehensive Dengue Serotype Detection

Abstract

Dengue fever, caused by the dengue virus (DENV), remains one of the most prevalent mosquito-borne diseases worldwide, significantly impacting tropical regions. Due to the existence of multiple serotypes (DENV 1-4), secondary infection with a different serotype significantly increases the risk of severe dengue, making serotype differentiation important. Expanding mosquito habitats due to climate change and rapid urbanization have increased global transmission, particularly in low-resource regions where laboratory-based diagnostics are limited. Current gold-standard methods, such as reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), require specialized equipment and centralized facilities, creating a need for accessible point-of-care diagnostics. This project aims to develop a low-cost, portable diagnostic tool that utilizes reverse transcription loop-mediated isothermal amplification (RT-LAMP) with a multiplex lateral flow assay (LFA) to detect and differentiate all four DENV serotypes. The amplified products will then be detected using streptavidin-coated gold nanoparticles (AuNPs) and DNA capture probes immobilized by high salt concentration on multiplex LFA strips, eliminating the need for antibodies. Initial testing with synthetic DNA samples demonstrated successful amplification of the DENV-2 serotype at 46,671 copies/reaction using colorimetric in around 45 minutes. Future work will include optimizing LAMP sensitivity and further validation with Fluorescent LAMP to determine the absolute limit of detection. A portable heating device has already been 3D printed to enable instrument-free amplification. This diagnostic tool has the potential to improve early diagnosis and outbreak monitoring in resource-limited settings.