Abstract

Circulating tumor DNA (ctDNA), a fraction of cell-free DNA (cfDNA) in the circulatory system, is released from tumor cells and thus carries tumor-specific genetic signatures. Using blood-derived ctDNA to detect somatic mutations has shown great value in guiding cancer targeted therapy. Isolation and detection efficiencies are the key factors affecting the performance of ctDNA detection. To optimize and standardize our clinical practice, in this study, we analyzed the isolation efficiency of four commercial cfDNA purification kits: QIAamp circulating nucleic acid kit, AmoyDx^® Circulating DNA kits, Microdiag^® circulating DNA isolation kit, and MagMAX cell-free DNA isolation kit; and the detection efficiency of two mainstream domestic EGFR gene mutation detection kits: MicroDiag EGFR gene mutation detection kit and Fluorometric real-time PCR Detection Kit for the analysis of EGFR gene mutations. Reference materials and plasma samples collected from lung cancer patients and healthy volunteers were used for the analysis. Our results showed that QIAamp circulating nucleic acid kit and Microdiag^® circulating DNA kit had the highest recovery rate (up to 21.25 ng/mL) for short DNA fragments of about 173 bp which is the peak length of ctDNA. For ctDNA detection, the MicroDiag^® EGFR gene mutation detection kit showed the highest detection rate and sensitivity for detecting EGFR mutations at a mutant frequency of 0.5%. This work provides a reliable choice of commercial kits for the clinical application of ctDNA.

摘要

循环肿瘤 DNA (ctDNA) 是循环系统中游离细胞 DNA (cfDNA) 的一部分，从肿瘤细胞中释放出来，因此带有肿瘤特异性遗传特征。使用血液来源的 ctDNA 检测体细胞突变在指导癌症靶向治疗方面显示出巨大价值。分离和检测效率是影响 ctDNA 检测性能的关键因素。为了优化和规范我们的临床实践，在本研究中，我们分析了四种商业 cfDNA 纯化试剂盒的分离效率：QIAamp 循环核酸试剂盒、AmoyDx ^®循环 DNA 试剂盒、Microdiag ^®循环 DNA 分离试剂盒和 MagMAX 无细胞 DNA 分离成套工具; 以及国内两种主流EGFR的检测效率基因突变检测试剂盒：用于EGFR基因突变分析的MicroDiag EGFR基因突变检测试剂盒和荧光实时PCR检测试剂盒。从肺癌患者和健康志愿者收集的参考材料和血浆样本用于分析。我们的结果表明，QIAamp 循环核酸试剂盒和 Microdiag ^®循环 DNA 试剂盒对 ctDNA 峰长约 173 bp 的短 DNA 片段的回收率最高（高达 21.25 ng/mL）。对于ctDNA检测，MicroDiag ^® EGFR基因突变检测试剂盒显示出最高的检测EGFR的检出率和灵敏度 突变频率为 0.5%。这项工作为 ctDNA 的临床应用提供了可靠的商业试剂盒选择。