通过复合评分响应面法对离子调控模式进行多维验证

Abstract

Divergent ion-regulation patterns are revealed: Mg 2+ -NH 4 + antagonism in DNA system vs. Mg 2+ -K + complementarity in RNA system. • A composite-score response surface strategy simultaneously optimizes speed, specificity, efficiency, and repeatability for LAMP/RT-LAMP assays. • Thermal stability and Michaelis–Menten analyses elucidate the underlying mechanisms: NH 4 + destabilizes Bst polymerase, while K + lowers the apparent Km. • The composite-score-optimized framework achieved a 3-fold (DNA) and a 10-fold (RNA) improvements in the lowest detectable concentration compared to a commercial RT-LAMP kit. 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: Mg 2+ and NH 4 + showed strong antagonism, whereas Mg 2+ 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. Download: Download high-res image (307KB) Download: Download full-size imag