DISCO-LAMP: a novel discontinuous LAMP assay for isothermal antigen detection

Proximity ligation assay (PLA) is a highly sensitive protein detection method in which DNA probes conjugated to affinity reagents are brought into proximity upon binding the same target molecule, thereby enabling greatly accelerated ligation of the DNA strands. The detection of the ligated DNA typically relies on PCR, which requires temperature cycling. In this study, we report on a novel discontinuous (DISCO)-LAMP assay that enables the wash-free detection of PLA products via loop-mediated isothermal amplification (LAMP). Due to the exponential amplification nature of LAMP, a careful balance between efficient amplification of the ligated full-length DNA and minimal background amplification from the individual constituent DNA fragment is essential but often challenging to achieve. After extensive template/primer design and assay optimization, DISCO-LAMP assay achieved a detection limit of 1 fM for the full-length DNA while maintaining minimal background amplification at 10 nM of each individual DNA fragment. When functionalized with specific affinity reagents, DISCO-LAMP detected Shiga toxin 2 (Stx2) with a limit of detection (LoD) of 100 fM as well as both Wuhan-1 and Omicron spike protein using a newly engineered Designed Ankyrin Repeat Protein (DARPin) D_S16 targeting a conserved epitope on the SARS-CoV-2 spike protein. We believe DISCO-LAMP represents a versatile and efficient LAMP-based technology that is readily adaptable for sensing diverse targets via PLA.