Multiplexed Detection of Pathogens Using Solid-Phase Loop-Mediated Isothermal Amplification on a Supercritical Angle Fluorescence Array for Point-of-Care Applications

Adaptations of new generation molecular techniques for multiplexed detection of pathogens are gaining interest in the field of point-of-care (POC) industry and onsite testing. Loop-mediated isothermal amplification (LAMP), an advanced molecular amplification technique, has proven promising due to its unique features that suits ideal for POC applications. However, application of LAMP for multiplexed detection of pathogens remains challenging because of the difficulty in the identification of specific LAMP amplicons that does not have a well-definite molecular size. In this study, we developed a solid-phase loop-mediated isothermal amplification (SP-LAMP) technique to address the challenge. Integration of LAMP with the supercritical angle fluorescence (SAF) micro-optic structures as a solid support (SS) in an array format enabled spatial separation of LAMP amplicons in a multiplexed configuration. Important parameters such as length of the SS primers, length of the primer-binding region, the effect of surface density of immobilized SS primers, and cross-reactivity among the primers of different targets were iteratively tested and optimized. With the combination of SP-LAMP and SAF techniques, it was possible to detect multiple pathogens that include Salmonella spp, Campylobater spp., Campylobacter coli, Campylobacter jejuni, avian influenza virus (AIV), and pan avian internal control (IC) under singleplex conditions. The multiplexing capacity of the SP-LAMP was demonstrated using AIV and IC with promising results. The success of SP-LAMP has opened a promising direction toward the development of a multiplex POC system for rapid detection of multiple pathogens.