Loop-mediated isothermal amplification (LAMP) has become a cornerstone of nucleic acid detection for clinical diagnostics, environmental monitoring, and point-of-care testing, offering rapid amplification with minimal equipment needs. Operating at a constant temperature, LAMP avoids the complexities of thermal cycling required by PCR, enabling easier field deployment. However, the method’s reliance on custom-designed sets of four to six primers per target complicates assay development, increases costs, and slows responsiveness to emerging pathogens and variants.
Efforts to expand LAMP’s versatility often introduce additional steps, such as ligation or multi-enzyme workflows, complicating use in low-resource settings. Multiplexing is particularly challenging due to the risk of primer–dimer formation and non-specific amplification when multiple sequence-specific primers are used. Current approaches to sequence larger target regions for downstream analysis require extensive hands-on time and specialized reagents, limiting the adaptability of LAMP for real-time surveillance and high-throughput diagnostics.
This technology introduces a universal LAMP system that utilizes chimeric primers composed of a 3′ region complementary to the target sequence fused to 5′ segments of a standardized LAMP template. In a single-pot isothermal reaction with standard LAMP primers (FIP, BIP, F3, B3), chimeric primers, and Bst 2.0 DNA polymerase, the system generates concatemeric amplicons embedding captured target sequences between F1 and B1 regions. Amplification can be monitored in real time using oligonucleotide strand displacement probes, and the resulting products are directly compatible with nanopore or sequencing-by-synthesis platforms for downstream sequence analysis.
This method eliminates the need for target-specific primer redesign for each assay, dramatically simplifying development and enabling rapid adaptation to new targets. Unlike traditional LAMP or rolling circle amplification systems that require DNA ligase or other accessory enzymes, this platform operates in a one-pot reaction with only Bst polymerase. Its design supports simultaneous multiplex detection using a universal primer set and enables both real-time monitoring and seamless integration with portable sequencing platforms for mutation detection and pathogen surveillance.