FRET-Based Assimilating Probe for Sequence-Specific Real-Time Monitoring of Loop-Mediated Isothermal Amplification (LAMP)

Isothermal nucleic acid amplification offers compelling opportunities for simple gene-based diagnostics, although the technologies for monitoring these reactions in real-time are still relatively undeveloped compared to PCR-specific probes. In this article, we describe the adaptation of an innovative FRET-based probe architecture for monitoring the isothermal LAMP reaction in real-time, allowing single-step, direct identification of different isolates of the agricultural pathogen Ralstonia solanacearum. The probe design is analogous to a two-component molecular zipper used for monitoring rolling circle amplification (RCA), in which a quenching oligonucleotide strand is displaced from a fluorescent strand by a strand-displacing polymerase during the reaction. Because the fluorescent strand is stably assimilated into the LAMP amplicon in our configuration, we use the nomenclature assimilating probe to describe the probe. Using these probes, we have demonstrated the ability to perform real-time, quantitative LAMP analysis of DNA, detecting fewer than ten gene copies. Probe compositions were optimized to prevent noticeable inhibition of the LAMP reaction, allowing observation of kinetics for different LAMP reactions and inference of primer design rules for accelerated detection. The sequence-specific nature of the assimilating probes offers compelling advantages over non-specific methods for real-time detection of LAMP amplification, including improved selectivity and the potential for multiplexed detection.