血细胞游离 DNA (cfDNA) 可以成为检测非小细胞肺癌 (NSCLC) 患者表皮生长因子受体 (EGFR) 突变的可靠新工具。然而，目前报道的 cfDNA 检测由于其灵敏度、稳定性或突变检出率方面的缺陷，在检测耐药突变方面作用有限。我们开发了一种基于古生球菌衍生的瓣状核酸内切酶 (Afu FEN) 的 DNA 增强扩增通过设计一对发夹探针与野生型 cfDNA 退火形成两个 5'-襟翼，允许 Afu FEN 对野生型 cfDNA 进行特异性切割，从而构建突变 cfDNA 系统。当优势野生型体细胞cfDNA片段被结构识别特异性的Afu FEN切割时，反应体系中突变cfDNA的比例大大富集。由于通过 PCR 扩增进一步增加了系统中突变 cfDNA 的数量，因此可以通过第一代测序轻松检测突变状态。在合成的野生型和 T790M EGFR DNA 片段的混合物中，我们的新检测方法仍然可以检测到 T790M fg 水平的突变具有非常高的敏感性。我们还测试了其在检测 NSCLC 患者临床样本中低变异等位基因频率 (VAF) 突变方面的性能。低 VAF（0.1 和 0.5%）的血浆 cfDNA 样本可以通过 DNA 增强扩增轻松检测到。该突变 cfDNA 增强扩增系统通过提供快速检测，是用于 NSCLC 早期筛查和个体化靶向治疗的有效工具。用于检测肿瘤耐药突变的、灵敏且经济的方法。© 2024 Walter de Gruyter GmbH，柏林/波士顿。

Blood cell-free DNA (cfDNA) can be a new reliable tool for detecting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. However, the currently reported cfDNA assays have a limited role in detecting drug-resistant mutations due to their deficiencies in sensitivity, stability, or mutation detection rate.We developed an Archaeoglobus fulgidus-derived flap endonuclease (Afu FEN)-based DNA-enhanced amplification system of mutated cfDNA by designing a pair of hairpin probes to anneal with wild-type cfDNA to form two 5'-flaps, allowing for the specific cleavage of wild-type cfDNA by Afu FEN. When the dominant wild-type somatic cfDNA fragments were cleaved by structure-recognition-specific Afu FEN, the proportion of mutated cfDNA in the reaction system was greatly enriched. As the amount of mutated cfDNA in the system was further increased by PCR amplification, the mutation status could be easily detected through first-generation sequencing.In a mixture of synthetic wild-type and T790M EGFR DNA fragments, our new assay still could detect T790M mutation at the fg level with remarkably high sensitivity. We also tested its performance in detecting low variant allele frequency (VAF) mutations in clinical samples from NSCLC patients. The plasma cfDNA samples with low VAF (0.1 and 0.5 %) could be easily detected by DNA-enhanced amplification.This system with enhanced amplification of mutated cfDNA is an effective tool used for the early screening and individualized targeted therapy of NSCLC by providing a rapid, sensitive, and economical way for the detection of drug-resistant mutations in tumors.© 2024 Walter de Gruyter GmbH, Berlin/Boston.