Browser-based design of loop-mediated isothermal amplification (LAMP) primer sets with template repeat masking and bisulphite support

Loop-mediated isothermal amplification (LAMP) requires four to six oligonucleotides to occupy six to eight defined regions of a target, with strict constraints on spacing and melting temperature. This makes manual design impractical; therefore, most users rely on a limited number of server-side design services. In this study, a browser-based LAMP primer set designer is presented that requires no installation or registration and does not transmit the target sequence. The tool generates six independent candidate lists per target and screens each candidate for melting temperature, linguistic complexity, simple sequence content, self-complementarity, and complementarity to existing user-owned oligonucleotides. The remaining candidates are then assembled into complete F3/F2/F1c-B1c/B2/B3 assays, optionally including loop primers. Any combination in which two members can form a stable duplex is first rejected by a fast combinatorial rule and then by the nearest-neighbor free energy of the most stable dimer that the assembled molecules can form at the reaction temperature. Regions of the template covered by dispersed repeats or low-complexity sequences are masked prior to design. The tool's yield, parameter sensitivity, and masking behavior are reported, and its output is compared with an archived PrimerExplorer V5 design on an identical target. Four four-primer LAMP assays for transgene screening, which were designed using an earlier version of the same principles and validated experimentally, are also described. The tool is freely available at https://primerdigital.com/tools/lamp.html.