Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting

Loop-mediated isothermal amplification (LAMP) has proven to be easier to implement than PCR for point-of-care diagnostic tests. However, the underlying mechanism of LAMP is complicated and the kinetics of the major steps in LAMP have not been fully elucidated, which prevents rational improvements in assay development. Here we present our work to characterize the kinetics of the elementary steps in LAMP and show that: (i) strand invasion / initiation is the rate-limiting step in the LAMP reaction; (ii) the loop primer plays an important role in accelerating the rate of initiation and does not function solely during the exponential amplification phase and (iii) strand displacement synthesis by Bst-LF polymerase is relatively fast (125 nt/s) and processive on both linear and hairpin templates, although with some interruptions on high GC content templates. Building on these data, we were able to develop a kinetic model that relates the individual kinetic experiments to the bulk LAMP reaction. The assays developed here provide important insights into the mechanism of LAMP, and the overall model should be crucial in engineering more sensitive and faster LAMP reactions. The kinetic methods we employ should likely prove useful with other isothermal DNA amplification methods.

Automated summary

Loop-mediated isothermal amplification (LAMP) is easier to implement than PCR for point-of-care diagnostic tests. This study characterizes the kinetics of the elementary steps in LAMP and shows that the rate-limiting step is strand invasion / initiation. The loop primer accelerates the rate of initiation and Bst-LF polymerase demonstrates fast and processive strand displacement synthesis. These findings contribute to a kinetic model that can be used to engineer more sensitive and faster LAMP reactions, with potential applications for other isothermal DNA amplification methods.