Kapadia, J. B.; N'Diaye, K. D. S.; Daoud, J.; Perreault, J.

An enzyme free nucleic acid amplification method based on toehold mediated strand displacement reaction (TMSDR) was evaluated under a variety of conditions with the aim of eliminating conventional purification steps and streamlining diagnostic workflows. By operating directly in lysis buffers, the TMSDR assay enhances target recovery and confers protection against nuclease degradation. Amplification performance was examined in the presence of diverse denaturing chemicals, lysis buffers, and sample matrices, including blood, saliva, wastewater, and soil, and the results demonstrated broad versatility and robust amplification under most conditions. The assay-maintained efficacy even in the presence of common PCR inhibitors, such as polyphenols in plant extracts, immunoglobulin G in blood, and complex constituents in environmental samples. Furthermore, a proof-of-concept assay targeting the 16S rRNA of Escherichia coli was established using specifically designed probe and displacer sequences, with specificity confirmed by the absence of amplification in mutated target controls. Collectively, these findings underscore the potential of the TMSDR assay as a highly adaptable and efficient alternative for rapid nucleic acid detection in point-of-care and field applications. This enzyme-free, purification-independent platform opens the door for rapid diagnostic development in resource-limited settings.