PO.CL01.11 · 临床研究
通过整合高体积cfDNA提取和液滴数字PCR突变检测提高液体活检的敏感性和可扩展性
Enhanced sensitivity and scalability in liquid biopsy through integrated high-volume cfDNA extraction and droplet digital PCR mutation detection
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：检测血浆中罕见的肿瘤来源变异既需要高效回收游离DNA（cfDNA），也需要高度定量的突变分析。传统提取方法受限于低投入量容量以及对较长片段的潜在偏好，这可能会损害对低频等位基因的敏感性。
方法：使用nRichDX Revolution cfDNA Max20 Kit（一种高体积磁珠系统）提取血浆cfDNA，然后与Qiagen QIAamp Circulating Nucleic Acid Kit进行比较。汇集的人血浆（1-15 mL，n=3）以及掺入1% EGFR E746_A750delELREA（COSM6223）和KRAS G12C（COSM516）突变gBlocks的模拟样本，均按照制造商方案处理。cfDNA浓度通过Qubit荧光法测量，突变分数丰度使用Bio-Rad QX600™ Droplet Digital™ PCR系统（ddPCR™）定量。在不同输入体积下评估线性、回收效率和重现性。
结果：nRichDX Revolution cfDNA Max20 Kit在1-15 mL血浆输入量范围内表现出线性cfDNA回收（R²=0.98），并可扩展至高达50 mL而无需重新浓缩或并行提取。当与Bio-Rad ddPCR系统联用时，突变分析始终能检测到1%的变异等位基因频率，与使用载体RNA的Qiagen QIAamp Circulating Nucleic Acid Kit相比，所测得的分数丰度高出15-30%。nRichDX工作流程保留了对突变检测至关重要的短cfDNA片段，改善了信噪比并增强了分析敏感性。重复变异性保持在10%以下，证实了在不同输入体积下稳健的精度。
结论：将nRichDX Revolution cfDNA Max20 Kit与Bio-Rad QX600 ddPCR系统整合，提高了基于cfDNA的液体活检应用的分析敏感性、回收准确性和可扩展性。通过保持cfDNA完整性并消除载体RNA干扰，该工作流程能够可靠地定量低频变异，支持早期检测、治疗监测和微小残留病灶评估方面的临床研究。
查看英文原文 English abstract
Introduction: Detecting rare tumor-derived variants in plasma requires both efficient recovery of cell-free DNA (cfDNA) and highly quantitative mutation analysis. Conventional extraction methods are limited by low input capacity and potential bias toward longer fragments, which can compromise sensitivity for low-frequency alleles.
Methods: Plasma cfDNA was extracted using the nRichDX Revolution cfDNA Max20 Kit, a high-volume magnetic-bead-based system, and then compared with the Qiagen QIAamp Circulating Nucleic Acid Kit. Pooled human plasma (1-15 mL, n = 3) and contrived samples spiked with 1% EGFR E746_A750delELREA (COSM6223) and KRAS G12C (COSM516) mutant gBlocks were processed per manufacturer's protocols. cfDNA concentration was measured by Qubit fluorometry, and mutation fractional abundance was quantified using the Bio-Rad QX600™ Droplet Digital™ PCR System (ddPCR™). Linearity, recovery efficiency, and reproducibility were evaluated across input volumes.
Results: The nRichDX Revolution cfDNA Max20 Kit demonstrated linear cfDNA recovery across 1-15 mL plasma inputs (R² = 0.98) and maintains scalability up to 50 mL without reconcentration or parallel extractions. When coupled with the Bio-Rad ddPCR System, mutation analysis consistently detected 1% variant allele frequencies, yielding 15-30% higher measured fractional abundance compared to the Qiagen QIAamp Circulating Nucleic Acid Kit, which uses carrier RNA.The nRichDX workflow preserved short cfDNA fragments critical for mutation detection, improving signal-to-background ratios and enhancing analytical sensitivity. Replicate variability remained below 10%, confirming robust precision across input volumes.
Conclusions: Integration of the nRichDX Revolution cfDNA Max20 Kit with the Bio-Rad QX600 ddPCR System enhances analytical sensitivity, recovery accuracy, and scalability for cfDNA-based liquid biopsy applications. By maintaining cfDNA integrity and eliminating carrier RNA interference, this workflow enables the reliable quantification of low-frequency variants, supporting clinical research in early detection, therapy monitoring, and minimal residual disease assessment.
利益披露 Disclosure
N. Jafari,
nRichDX Employment.
A. Partner,
Bio-Rad Laboratories, Inc Employment.
N. Mehmet,
Bio-Rad Laboratories, Inc Employment.
N. Kumar,
BioRad Employment.
P. Pal,
Bio-Rad Laboratories, Inc Employment.
J. Saenz,
nRichDX Employment.
C. Hernandez,
nRichDX Employment.
D. Cedeno,
nRichDX Employment.
C. Van Dieren,
nRichDX Employment.
M. Saidian,
nRichDX Employment.