Enzyme-free temperature resilient amplification assay with toehold stem-loop probe†

Toehold mediated strand displacement reaction (TMSDR) offers a rapid, enzyme-free amplification strategy, providing advantages over traditional methods like RT-PCR, and RT-LAMP. Optimizing TMSDR can significantly enhance sensitivity in point-of-care biosensor applications for target nucleic acid detection. However, achieving optimal performance requires meticulous probe design and stringent quality control. We developed a TMSDR-based system targeting a specific SARS-CoV-2 RNA sequence through testing multiple fluorophore–quencher labeled DNA probes. Following optimization, a probe with a strategically designed: stem, loop, and optimized toehold length emerged as the most effective candidate. Displacer sequence optimization further enhanced amplification efficiency. Ensuring probe purity is crucial, as impurities elevated background noise and diminished sensitivity. This work underscores the importance of rigorous probe quality in achieving reliable and sensitive TMSDR-based viral RNA detection, paving the way for robust point-of-care diagnostic tools.