A universal kinetic framework for quantitative isothermal amplification governed by polymerase speed, amplicon size,...

Area of Science:

• Molecular Biology
• Biophysics
• Biochemistry

Background:

• Isothermal exponential amplification (IEA) has historically relied on empirical optimization due to the absence of predictive, first-principles models.
• This lack of theoretical understanding has limited the quantitative potential and broad applicability of IEA techniques.

Purpose of the Study:

• To establish a universal kinetic framework for isothermal exponential amplification (IEA).
• To define IEA efficiency using fundamental physical parameters and provide a unified explanation for various IEA mechanisms.

Main Methods:

• Developed a kinetic model based on polymerase extension rate (Se), amplicon size (Sa), and primer-template binding efficiency (ξ).
• Utilized mathematical analysis, including Taylor expansion, to demonstrate the kinetics of loop-mediated isothermal amplification (LAMP).
• Experimentally confirmed predictions regarding LAMP product heterogeneity as a Poisson process.

Main Results:

• The apparent doubling time ($T$) is modeled as $T = \frac{{{{S}_a}}}{{\xi \cdot {{S}_e}}}$, unifying IEA efficiency across LAMP, SDA, RPA, and HDA.
• Demonstrated that complex LAMP kinetics are structurally isomorphic to simple exponential growth.
• Confirmed that LAMP product heterogeneity follows a Poisson process, enabling accurate quantification under various conditions, including viral detection in wastewater.

Conclusions:

• The established kinetic framework provides a predictive model for IEA, moving beyond empirical optimization.
• The model enables robust viral quantification and offers a blueprint for optimizing IEA by tuning Se, Sa, and ξ.
• This work bridges fundamental enzymology with practical point-of-care applications.