Blocker Primer-Assisted LAMP Coupled with Pyrococcus furiosus Argonaute Enables Specific and Sensitive Detection of EGFR Mutations in NSCLC

Abstract

Sensitive and specific detection of low-frequency mutations in circulating tumor DNA (ctDNA) remains a major challenge because mutant alleles typically constitute less than 1% of the total cell-free DNA in plasma. Herein, we present a blocker primer-assisted loop-mediated isothermal amplification (LAMP) assay coupled with Pyrococcus furiosus Argonaute (PfAgo) for detection of EGFR mutations associated with nonsmall cell lung cancer (NSCLC). A blocker primer was incorporated into the LAMP reaction to selectively suppress wild-type (WT) amplification and enrich the mutant-derived amplicons. The mutant amplicons were subsequently recognized and cleaved by PfAgo. The resulting cleavage fragments acted as secondary guides to trigger cleavage of fluorogenic probes, enabling cascade signal amplification. The assay detected the EGFR L858R, T790 M and 19del at mutant allele frequencies as low as 0.05% in a strong WT background (1 × 106 copies/μL total DNA) and reliably detected target mutations in lung cancer cell lines. In clinical plasma samples, the assay detected EGFR L858R with 84.62% sensitivity and 100% specificity relative to next-generation sequencing (NGS). These results demonstrate that the blocker primer-assisted LAMP coupled with PfAgo assay is a specific and sensitive approach for ctDNA mutation analysis and shows promise for liquid biopsy applications.