Non-spectroscopic multiplex molecular diagnostic platform based on loop-mediated isothermal amplification (LAMP) with specially designed primers

Abstract

Herein, we developed a novel multiplex molecular diagnostic platform based on the integration of loop-mediated isothermal amplif ication (LAMP) and retroreflective signaling. LAMP is a striking method that allows for rapid and specific gene amplif ication at a constant temperature, simplifying the need for equipment, such as a thermocycler. However, it sti l l faces challenges when it comes to post-amplif ication analysis. This is due to the intricate optical setup it demands, involving a complex arrangement of optical components. To minimize the se requirements, we util ized retroreflective signaling which can induce signals from non-spectral l ight source. This approach with LAMP eliminates the need for sophisticated equipment and makes the molecular diagnostics accessible. In this study, we introduced specially designed primer sets to generate double - labeled amplicons. These possess small molecule antigen and biotin at each end, facil itating their detection in the same manner as the sandwich-type assay. Multiplex sensing with avidin-conjugated RJPs (A-RJPs) could be available in this system by applying different pairs of small molecule antigens and capture molecule while maintaining biotin. We introduced FITC and Texas Red as small molecule antigens for the invA of Salmonella and tetracycline resistance gene ( tet), respectively. In addition, for the validation of the chip uti l ized in the assay, a control channel was integrated into the multi-sensing chip. Therefore, it allows the simultaneous detection of multiple targets and validation of the results using only o ne type of optical probe. Based on the developed platform, a highly sensitive and selective quantif ication of antibiotic-resistant Salmonella was successfully performed with a detection l imit of 1 colony-forming units (CFU).