Van der Snickt, T.; Ailliet, S.; Bassini, S.; Peymen, A.; Colaker, A.; Cadoni, E.; Valverde, A.; Daems, E.; Madder, A.; De Wael, K.; Mestdagh, P.; SOCan consortium,

Enzyme-free isothermal amplification methods offer a promising alternative to enzymatic assays for nucleic acid detection, particularly in low-resource settings. The nonenzymatic isothermal strand displacement and amplification (NISDA) assay was recently introduced as a highly sensitive, enzyme-free detection strategy. Here, we attempted to replicate its reported performance. Despite extensive testing, we failed to replicate the reported sensitivity and could only produce detectable signals at extremely high target concentrations, specifically at or above one hundred billion copies per microliter. Our results highlight the critical importance of independent validation in the development of nonenzymatic diagnostic assays.