Abstract
Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne viruses that cause severe health problems upon infection, necessitating timely and accurate diagnostic testing for effective prevention and control of their transmission. In this study, a novel multiplex nucleic acids detection method was introduced based on ligation-based universal primer and probe loop-mediated isothermal amplification (LUPP-LAMP) for the simultaneous detection and genotyping of DENV and ZIKV. By employing ligation of universal primers and probes using splint DNA ligase, LUPP-LAMP simplifies primer design, eliminates primer dimer formation, and achieves high sensitivity and specificity at low temperatures, with a detection limit of 10 copies/reaction. The robustness of this strategy allows for precise differentiation even at low concentrations and in cases of co-infection, which is crucial for addressing early diagnostic challenges due to similar symptoms. LUPP-LAMP is further adapted for point-of-care testing (POCT) by integrating lateral flow test strips and fluorescence imaging, offering rapid and user-friendly results in resource-limited settings. This method is also applied to DENV genotyping (serotypes 1-4) using melting curve analysis (MCA) in cases of co-infection, aiding in epidemiological surveillance and intervention strategies. In summary, LUPP-LAMP represents a significant advancement in the detection and genotyping of DENV and ZIKV, offering a versatile tool for laboratory and POCT applications, thereby enhancing public health outcomes in the control of mosquito-borne diseases.