High-temperature ssBP-LAMP breaks the barrier of nonspecific amplification and unlocks the full potential of LAMP diagnostics

Loop-mediated isothermal amplification (LAMP) is a highly attractive nucleic acid detection method for decentralized molecular diagnostics, but its broader adoption has been limited by unpredictable nonspecific amplification (NSA). Here, we dissect the fundamental determinants of LAMP specificity and present a new high-temperature ssBP-LAMP technique to suppress NSA without compromising assay sensitivity or speed. Ubiquitous bioinformatic tools for prediction and design of primer sets and their characteristics were employed to generate a panel of 21 primer sets targeting diverse bacterial, viral, fungal, and human targets. Fluorometric ssBP-LAMP was implemented with a series of coordinated modifications, from increased temperature (only possible owing to a novel, engineered thermostable strand displacement Bst polymerase enzyme) to fine-tuning of additives and auxiliary enzymes. Finally, proof-of-concept validation using both fluorometric and colorimetric approaches for ssBP-LAMP was performed, and their resistance to common inhibitors of molecular diagnostics was evaluated. We show for the first time that primer design alone is insufficient to ensure specificity, and bioinformatic predictions are insufficient predictors of efficiency and, more importantly, of specificity, even when using elevated reaction temperatures. This suggests that NSA frequently originates during the thermal ramp-up phase, before the assay reaches its operational temperature. To address this, we employed a thermolabile single-stranded DNA-binding protein (ssBP) from bacteriophage T7, which transiently sequesters primers during temperature ramp-up. When combined with conventional LAMP primers and elevated assay temperatures (≥ 68 °C), ssBP consistently suppressed NSA across all tested targets. This approach translated directly to both liquid and lyophilized master mixes, enabling highly specific ssBP-LAMP detection by real-time fluorescence and colorimetry. These formulations retained robust sensitivity (down to 10–100 copies/reaction for real-time detection), eliminated false positives, and exhibited tolerance to clinically relevant inhibitors such as plasma, hemoglobin, saliva, and urine, especially in real-time fluorescence detection. Together, the results establish high-temperature ssBP-LAMP as a rapid (< 15 min), specific, inhibitor-tolerant, and highly adaptable molecular diagnostic technology that overcomes key limitations of conventional LAMP while achieving analytical performance comparable to qPCR, with broad relevance for both clinical and point-of-care testing.