人体细胞中不可避免地会积累基因突变。在生理和病理过程中尽早检测突变具有重要意义。然而，由于存在丰富的野生DNA背景，检测患者低拷贝肿瘤DNA（ctDNA）中的突变具有挑战性。本研究引入了人工发夹（artificial hairpins）到扩增子中，通过与引物竞争的方式在低等位基因频率（VAF）下富集罕见突变，创造了一种新的策略，被称为发夹竞争扩增（hairpin competition amplification，HCA）系统。本文首次研究了在高温PCR系统中人工发夹对引物结合的影响，为HCA的优化铺平了道路。HCA与以前报道的使用人工发夹抑制野生型DNA扩增的方法不同，后者使用5-外切酶负链型聚合酶，在非对称PCR后通过熔解曲线分析来读出结果。HCA针对六种不同变异体，进行了HCA qPCR和HCA Sanger测序，能够富集0.1%或0.01%等低等位基因频率的突变DNA。HCA在多重反应和温度稳定性方面显示出优势。在对12例肺癌ctDNA样本和16例甲状腺癌FNA DNA样本进行基因状态分析时，与ddPCR和商业ARMS试剂盒相比，HCA显示出了100%的一致性。HCA qPCR和Sanger测序具有高灵敏度和温度稳定性，能够富集低丰度的突变体，为罕见变异性疾病的精准诊断和治疗提供了一种新型有效工具。

Gene mutations are inevitably accumulated in cells of the human body. It is of great significance to detect mutations at the earliest possible time in physiological and pathological processes. However, genotyping low-copy tumor DNA (ctDNA) in patients is challenging due to abundant wild DNA backgrounds. One novel strategy to enrich rare mutations at low variant allele fractions (VAFs) with quantitative polymerase chain reaction (qPCR) and Sanger sequencing was contrived by introducing artificial hairpins into amplicons to compete with primers, coined as the hairpin competition amplification (HCA) system. The influence imposed by artificial hairpins on primer-binding in a high-temperature PCR system was investigated for the first time in this work, paving the way for the optimization of HCA. HCA differs from the previously reported work in which hairpins are formed to inhibit extension of wild-type DNA using 5-exonuclease-negative polymerase, where the readout is dependent on melting curve analysis after asymmetric PCR. Targeted at six different variants, HCA qPCR and HCA Sanger-enriched mutant DNA at VAFs as low as 0.1 or 0.01% were performed. HCA demonstrated advantages in multiplex reaction and temperature robustness. In profiling gene status from 12 lung cancer ctDNA samples and 16 thyroid cancer FNA DNA samples, HCA demonstrated a 100% concordance rate compared to ddPCR and commercial ARMS kit. HCA qPCR and Sanger sequencing can enrich low-abundance variants with high sensitivity and temperature robustness, presenting a novel and effective tool for precision diagnosis and treatment of rare variant diseases.