PO.CL01.11 · 临床研究
通过3分钟捕获和5分钟纯化流程后,采用qPCR评估uProcess在缓冲液和尿液中检测癌基因序列的能力
Evaluation of uProcess for detection of oncogene sequences in buffer and urine by qPCR following 3-minute capture and 5- minute purification procedure
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
存在于血液和尿液中的游离DNA(cfDNA)和循环肿瘤DNA(ctDNA),已被牢固确立为诊断各类癌症及确定治疗方案的关键分析物。cfDNA中的原始序列和表观遗传标志物是癌症筛查和监测的关键。然而,筛查和长期监测都存在两大局限——检测灵敏度和个体参与度。即便NGS、ddPCR及其他方法持续改进,它们仍需大量cfDNA才能可靠地检测到罕见的ctDNA片段。例如,单次BioRad ddPCR反应可分析1 ug短片段cfDNA,但单管血液可能仅产出约10 ng的量,从而将潜在灵敏度削减100倍。此外,参与度通常是筛查和长期监测的一大严重障碍。历史上,这一直是主要障碍,尤其对筛查而言。前往诊所、静脉采血的疼痛/困难以及患者对此的抗拒都限制了参与度。尿液提供了一种更易获取的替代方案,但ctDNA浓度较低,且当前基于尿液的cfDNA/ctDNA采集方法尚未达到高灵敏度检测所需的产量。在0.001%水平上进行检测需要1 ug的cfDNA。在此,我们测试并优化了一套专为家庭尿液过滤设计的系统(uProcess);此前已证明该系统能通过邮寄式滤芯从200 mL尿液中捕获超过2 ug的cfDNA。与以往版本相比,我们将洗脱体积缩小了5倍,从2.5 ml降至0.5 ml,从而为下游处理提供浓缩样本。我们证明改进后的设备能在5分钟内高效地将缓冲液或尿液中的癌基因序列浓缩至小洗脱体积中,而典型流程需要90-120分钟。随后我们通过qPCR检测到KRAS和TP53癌基因,凸显该方法所产生的ctDNA适用于下游应用。综上所述,这些发现支持uProcess在显著扩大cfDNA/ctDNA检测可及性、提高灵敏度、缩短cfDNA纯化时间以及克服癌症筛查或监测长期存在的障碍方面的潜力。
查看英文原文 English abstract
Cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA), found in blood and urine, have firmly established themselves as key analytes for the diagnosis of various cancers and defining therapy. Primary sequence and epigenetic markers within cfDNA are key in screening and monitoring for cancer. However, both screening and long-term monitoring have two major limitations - test sensitivity and individual participation. Even as NGS, ddPCR, and other methods continue to improve, they require large amounts of cfDNA to reliably detect rare ctDNA fragments. For example, a single BioRad ddPCR reaction can analyze 1 ug of short cfDNA, but a single tube of blood might yield on the order of 10ng, thus cutting potential sensitivity 100x. Additionally, participation is typically a severe impediment to screening and long-term monitoring. Historically, this has been a major obstacle, especially for screening. Travel to clinics, pain/difficulty of phlebotomy, and patient aversion to it limit participation. Urine offers a far more accessible alternative, but ctDNA concentrations are lower and current urine-based cfDNA/ctDNA collection methods have yet to achieve yields adequate for highly sensitive testing. A ug of cfDNA is required for detection at the 0.001% level. Herein, we test and optimize a system (uProcess) designed for at-home urine filtration; it was previously demonstrated to capture over 2 ug of cfDNA from 200 mL of urine on a mail-in filter cartridge. Compared to previous versions, we have reduced the elution volume 5-fold from 2.5 to 0.5 mls, allowing concentrated samples for downstream processing. We demonstrated that the improved device can efficiently concentrate oncogene sequences, in buffer or urine, into a small elution volume in under five minutes vs. typical procedures taking 90-120 minutes. We then detected KRAS and TP53 oncogenes by qPCR, underscoring that the method produces ctDNA suitable for downstream usage. Together, these findings support the potential of uProcess to significantly expand access to cfDNA/ctDNA testing, improve sensitivity, shorten cfDNA purification time, and overcome longstanding barriers to cancer screening or monitoring.
利益披露 Disclosure
F. E. Taub,
aiGENE, Inc Employment, Stock, Stock Option, Patent, Other Intellectual Property.
S. Pearson,
aiGENE, Inc. Employment.
S. E. M. Wright,
aiGENE, Inc. Independent Contractor.
C. Neff,
aiGENE, Inc. Independent Contractor.