(228bu) Loop-Mediated Isothermal Amplification (LAMP): An Insight into Reaction Mechanism and Application in Point-of-Care Diagnostics

Analysis of nucleic acids is an essential part of both qualitative and quantitative diagnostics. Although polymerase chain reaction (PCR) based diagnostic techniques are well validated, the associated turnaround time, complex instrumentation and thermal cycling of reagents makes it suboptimal for point-of-care applications. Loop-mediated isothermal amplification (LAMP) is an alternate nucleic acid amplification method offering simple and cost-effective diagnosis of infectious diseases by eliminating thermal cycling and tedious sample preparation steps. It uses auto-cycling strand displacement synthesis with the aid of 6 specific primers. However, the intermediate reaction products are intricate stem loop DNA constructs (DNA hairpins), which may or may not contain the target sequence. This often makes LAMP a semi-quantitative analysis method suitable for yes/no type detection.

Although LAMP is increasingly used for diagnostic testing, current softwares for LAMP are limited to primer design, and unlike PCR, there is no known mechanistic description of the complete LAMP reaction. Here we present a kinetic model that explores all reaction intermediates and their evolution in respective reaction pathways. The kinetic parameters are estimated from literature and statistical mechanics calculation. The agent based model predicts around 15 side products along with the double loop DNA structure responsible for the autocatalytic amplification reaction. Some of these side products without the target sequence can lead to misrepresentation of the true amplification signal rendering LAMP as a semi-quantitative technique. The fundamental insights gained from our model are then used to determine optimal reaction parameters and efficient primer design. We leverage our new understandings to demonstrate multiplexed assay detection via the quencing of unincorporated amplification of signal reporters (QUASR) technique, enabling us to detect bright, unambiguous fluorescence signals with a simple smartphone based fluorimeter for point of care applications.

Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energyâ??s National Nuclear Security Administration under contract DE-AC04-94AL85000.