ICR-LAMP: A one-pot isothermal assay enabled by transient spatial separation and dynamic ICR for high-specificity SNP...

Single nucleotide polymorphisms (SNPs) serve as critical genetic markers associated with disease susceptibility, drug response, and pathogen evolution. However, most existing methods fail to simultaneously satisfy high specificity, sensitivity, and operational simplicity, limiting their practical application in decentralized settings. To address this gap, we developed a novel isothermal SNP genotyping approach named ICR-LAMP by integrating loop-mediated isothermal amplification (LAMP) and invasive cleavage reaction (ICR). We proposed an original "transient spatial separation" strategy to resolve the incompatibility between dynamic LAMP primer extension and static ICR structure formation, thereby realizing one-tube isothermal detection. Additionally, we identified a dynamic ICR cleavage mechanism that further enhances the exceptional single-nucleotide discrimination performance. After systematic optimization, ICR-LAMP achieved a detection limit of 10 copies/μL within 40 min and enabled unambiguous genotyping using a single probe based on endpoint fluorescence. Validation with four clinically significant SNPs (ABCB1 C3435T, CYP2C19∗2 G681A, MTHFR C677T, ALDH2 G1510A) demonstrated its outstanding clinical diagnostic performance. A duplex ICR-LAMP system was further developed, revealing its strong potential for high-throughput multiplex analysis and greatly improving detection efficiency. In summary, with the merits of simplicity, rapidity, low cost, and robustness, ICR-LAMP provides a universal and reliable platform for SNP genotyping in decentralized settings.