建立和验证一种超灵敏的qPCR方法来鉴定和定量游离DNA中的EGFR T790M。

IF 1.9 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS Bioanalysis Pub Date : 2025-01-01 Epub Date: 2025-01-15 DOI:10.1080/17576180.2025.2451527

Shenglei Yuan, Nan Jia, Guofu Lu, Jinping Lai, Wenzhong Liang, Lan Li, Chenpu Zhang, Jianbo Diao

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引用次数: 0

摘要

背景：循环肿瘤DNA （ctDNA）是一种很有前景的肿瘤预后和药物开发生物标志物。ctDNA检测方法面临的一个主要挑战是如何准确、灵敏地将ctDNA与高度相似但丰度更高的野生型DNA区分开来。方法：建立了一种检测EGFR T790M突变的超灵敏qPCR方法——三重富集扩增突变PCR （TEAM-PCR）。结果：EGFR T790M在存在106个野生型拷贝的情况下，在25-106拷贝/反应的检测范围内被定量。该方法按照基本生物分析指南进行了充分验证，检出限（LOD）为5拷贝/反应。结论：本研究建立并验证了一种基于qpcr的EGFR T790M突变检测策略，具有超高的灵敏度和可靠性。

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Development and validation of an ultrasensitive qPCR method to identify and quantify EGFR T790M in cell-free DNA.

Background: Circulating tumor DNA (ctDNA) is a promising biomarker for cancer prognosis and drug development. A major challenge in the ctDNA determination method is discriminating ctDNA from highly similar but significantly more abundant wild-type DNA sensitively and accurately.

Method: An ultrasensitive qPCR method termed Triple Enrichment Amplification of Mutation PCR (TEAM-PCR) was developed to detect EGFR T790M mutation.

Results: EGFR T790M was quantified over the assay range of 25-10⁶ copies/reaction in the presence of 10⁶ wild-type copies. This method was fully validated following the essential bioanalysis guidance, with the limit of detection (LOD) being five copies/reaction.

Conclusion: This study established and validated a qPCR-based strategy to detect EGFR T790M mutation with ultra-high sensitivity and reliability.

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来源期刊

Bioanalysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

3.30

自引率

16.70%

发文量

审稿时长

2 months

期刊介绍： Reliable data obtained from selective, sensitive and reproducible analysis of xenobiotics and biotics in biological samples is a fundamental and crucial part of every successful drug development program. The same principles can also apply to many other areas of research such as forensic science, toxicology and sports doping testing. The bioanalytical field incorporates sophisticated techniques linking sample preparation and advanced separations with MS and NMR detection systems, automation and robotics. Standards set by regulatory bodies regarding method development and validation increasingly define the boundaries between speed and quality. Bioanalysis is a progressive discipline for which the future holds many exciting opportunities to further reduce sample volumes, analysis cost and environmental impact, as well as to improve sensitivity, specificity, accuracy, efficiency, assay throughput, data quality, data handling and processing. The journal Bioanalysis focuses on the techniques and methods used for the detection or quantitative study of analytes in human or animal biological samples. Bioanalysis encourages the submission of articles describing forward-looking applications, including biosensors, microfluidics, miniaturized analytical devices, and new hyphenated and multi-dimensional techniques. Bioanalysis delivers essential information in concise, at-a-glance article formats. Key advances in the field are reported and analyzed by international experts, providing an authoritative but accessible forum for the modern bioanalyst.