Research Centre for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, Zhejiang Province, 310027, China; College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang Province, 310058, China.
Department of Clinical Pharmacology, Key Laboratory of Clinical Cancer Pharmacology and Toxicology Research of Zhejiang Province, Affiliated Hangzhou First People's Hospital, Cancer Center, Zhejiang University School of Medicine, Hangzhou, 310006, China.
Talanta. 2022 Jan 15;237:122977. doi: 10.1016/j.talanta.2021.122977. Epub 2021 Oct 16.
Nucleic acid extraction and purification before amplification is considered an essential step for nucleic acid amplification testing. However, this may cause losses or introduce errors that can lead to inaccurate results, especially when using samples with a small nucleic acid concentration. Here, we developed a direct digital chip that enabled us to detect nucleic acid without DNA extraction and purification. We have developed a self-priming liquid-dispensing digital PCR chip that does not require any external power. This is a robust anti-evaporation digital PCR chip with fast sampling and accurate quantification performance. Using this chip, we have established an on-chip direct nucleic acid amplification method that does not require nucleic acid extraction and purification for liquid biopsy samples. In order to verify the feasibility of this chip for clinical samples, we detected the EGFR T790M mutation from plasma. Results showed that EGFR T790M mutation could be detected with an accuracy of 100% and a sensitivity of 0.01%. Without nucleic acid extraction and purification, the assay avoids complex pre-processing, thus saving time and achieving precise quantification. We expect our direct digital PCR chip to have practical applications in diagnosis, screening, and research, especially in resource-deprived regions.
在扩增之前进行核酸提取和纯化被认为是核酸扩增检测的一个必要步骤。然而，这可能会导致损失或引入错误，从而导致结果不准确，特别是在使用核酸浓度较小的样本时。在这里，我们开发了一种直接数字芯片，使我们能够在不进行 DNA 提取和纯化的情况下检测核酸。我们已经开发出一种无需外部电源的自启动液滴分配数字 PCR 芯片。这是一种强大的抗蒸发数字 PCR 芯片，具有快速采样和准确定量性能。使用该芯片，我们建立了一种用于液体活检样本的芯片上直接核酸扩增方法，无需进行核酸提取和纯化。为了验证该芯片对临床样本的可行性，我们从血浆中检测了 EGFR T790M 突变。结果表明，该芯片能够以 100%的准确率和 0.01%的灵敏度检测 EGFR T790M 突变。无需核酸提取和纯化，该检测方法避免了复杂的预处理，从而节省了时间并实现了精确的定量。我们期望我们的直接数字 PCR 芯片在诊断、筛查和研究中具有实际应用价值，特别是在资源匮乏的地区。
