Multi-dimensional validation of ion-regulation patterns via composite-score RSM

The ionic requirements of loop‑mediated isothermal amplification (LAMP) and its reverse‑transcription variant (RT‑LAMP) remain poorly understood, especially regarding how the introduction of reverse transcriptase reshapes the ionic environment. Using monkeypox virus DNA and parainfluenza virus type 1 RNA as models, we systematically compared the ion‑regulation logic of the single‑enzyme (Bst‑only) and dual‑enzyme (Bst + reverse transcriptase) systems. We employed a stepwise experimental strategy to uncover ion‑response patterns, followed by mechanistic validation via protein thermal stability and Michaelis–Menten kinetic analyses. The Bst‑only (with MPXV DNA) and Bst+RT (with PIV1 RNA) systems examined in this study exhibited clearly distinguishable ion‑regulation patterns: Mg2+ and NH4+ showed strong antagonism, whereas Mg2+ and K+ displayed functional complementarity in the RNA system. These divergent patterns were further validated under a composite‑score framework, and the resulting optimized conditions outperformed a commercial RT‑LAMP kit by 3‑fold (DNA) and 10‑fold (RNA) in lowest detectable concentration. Collectively, these findings establish a paradigm for mechanism‑guided buffer optimization and provide a foundation for rational design of robust diagnostic assays.