The detection of multiple single nucleotide polymorphisms (SNPs) of circulating tumor DNA (ctDNA) is still a great challenge. In this study, we designed enzyme-assisted nucleic acid strand displacement amplification combined with high-performance liquid chromatography (HPLC) for the simultaneous detection of three ctDNA SNPs. First, the trace ctDNA could be hybridized to the specially designed template strand, which initiated the strand displacement nucleic acid amplification process under the synergistic action of DNA polymerase and restriction endonuclease. Then, the targets would be replaced with G-quadruplex fluorescent probes with different tail lengths. Finally, the HPLC-fluorescence assay enabled the separation and quantification of multiple signals. Notably, this method can simultaneously detect both the wild type (WT) and mutant type (MT) of multiple ctDNA SNPs. Within a linear range of 0.1 fM–0.1 nM, the detection limits of BRAF V600E-WT, EGFR T790M-WT, and KRAS 134A-WT and BRAF V600E-MT, EGFR T790M-MT, and KRAS 134A-MT were 29, 31, and 11 aM and 22, 29, and 33 aM, respectively. By using this method, the mutation rates of multiple ctDNA SNPs in blood samples from patients with lung or breast cancer can be obtained in a simple way, providing a convenient and highly sensitive analytical assay for the early screening and monitoring of lung cancer.

中文翻译：

---

链置换扩增结合液相色谱法检测循环肿瘤 DNA 的单核苷酸多态性

循环肿瘤DNA（ctDNA）的多个单核苷酸多态性（SNP）的检测仍然是一个巨大的挑战。在本研究中，我们设计了酶辅助核酸链置换扩增结合高效液相色谱（HPLC）来同时检测三个ctDNA SNP。首先，微量ctDNA可以与专门设计的模板链杂交，在DNA聚合酶和限制性内切酶的协同作用下启动链置换核酸扩增过程。然后，目标将被具有不同尾长的 G 四链体荧光探针替换。最后，HPLC 荧光测定能够分离和量化多个信号。值得注意的是，该方法可以同时检测多个ctDNA SNP的野生型（WT）和突变型（MT）。在 0.1 fM–0.1 nM 的线性范围内，BRAF V600E-WT、EGFR T790M-WT 和 KRAS 134A-WT 以及 BRAF V600E-MT、EGFR T790M-MT 和 KRAS 134A-MT 的检测限分别为 29、31分别是上午 11 点、22 点、29 点和 33 点。通过该方法，可以简单地获得肺癌或乳腺癌患者血液样本中多个ctDNA SNP的突变率，为肺癌的早期筛查和监测提供便捷、高灵敏度的分析方法。