MRPAND: A one-tube multiplex ago-mediated nucleic acid detection platform for visualized EGFR genotyping in point-of-care settings.

Current nucleic detection techniques suffer from prolonged timelines, high false-positive amplification, and poor point-of-care testing (POCT) compatibility, especially for multiplex assays. This highlights an urgent need for efficient diagnostics, particularly for the Epidermal Growth Factor Receptor (EGFR), a critical biomarker for non-small cell lung cancer (NSCLC) assessment, therapy, and postoperative monitoring. In this study, we established a one-tube detection platform, termed MRPAND, that integrates Multiplex Recombinant Polymerase Amplification (RPA) with Pyrococcus furiosus Argonaute-mediated Nucleic Acid Detection (PAND) for the identification of EGFR mutations. Capitalizing on the cleavage principle of PfAgo, the system enables simultaneous discrimination of two distinct mutations with high specificity. By integrating RPA with a lateral flow strip (LFS), the system achieves a sensitivity of 1 copy/μL with naked-eye visual readout, requires no sophisticated instrumentation, and is therefore well-suited for POCT applications. We further validated the clinical applicability of MRPAND using clinical samples, demonstrating its capability to identify EGFR mutations. These results underscore the potential of MRPAND as a promising diagnostic tool in lung cancer care.