Wu Wenshuai, Wu Fengtian, Zhang Shan, Ding Xiong, Zhang Tao, Yang Ying, Mu Ying
Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, 310027, PR China; College of Life Sciences, Zhejiang University, Hangzhou, 310058, PR China.
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310058, PR China.
Talanta. 2020 Dec 1;220:121426. doi: 10.1016/j.talanta.2020.121426. Epub 2020 Jul 21.
Circulating tumor DNA (ctDNA) is a promising biomarker for tumor genotyping and therapy monitoring. Herein, we developed a digital PCR chip with embedded microwell and bidirectional partition network for highly sensitive ctDNA analysis. The embedded microwell contributes to increasing microreaction density (up to 7000 microwells/cm) and reducing evaporation during amplification. The bidirectional partition network can achieve fast and random distribution of targets, ensuring the precise quantification of nucleic acid. We used plasmids, artificial samples and 32 clinical blood samples from non-small cell lung cancer patients to test the performance of this platform. The results demonstrated that our chip has not only comparable quantification performance to commercial counterpart but also the ability to detect EGFR mutations with as low as 0.01% mutation rate and 20 alter molecules in 27 ng genomic DNA. The identification of EGFR mutations in plasma using developed chip exhibited 85.71% sensitivity and 94.44% specificity for L858R mutation and 100% sensitivity and 86.96% specificity for T790 M mutation. Moreover, the monitoring of mutant allele in plasma was accomplished in this work. In conclusion, the developed chip has a potential in lung tumor genotyping and therapy monitoring for precision medicine, even other tumors.
循环肿瘤DNA（ctDNA）是一种用于肿瘤基因分型和治疗监测的有前景的生物标志物。在此，我们开发了一种带有嵌入式微孔和双向分区网络的数字PCR芯片，用于高灵敏度的ctDNA分析。嵌入式微孔有助于提高微反应密度（高达7000个微孔/平方厘米）并减少扩增过程中的蒸发。双向分区网络可以实现靶标的快速随机分布，确保核酸的精确定量。我们使用质粒、人工样本和32例非小细胞肺癌患者的临床血液样本测试了该平台的性能。结果表明，我们的芯片不仅具有与商业同类产品相当的定量性能，而且能够检测突变率低至0.01%且在27 ng基因组DNA中有20个变异分子的EGFR突变。使用开发的芯片对血浆中的EGFR突变进行鉴定，对于L858R突变表现出85.71%的灵敏度和94.44%的特异性，对于T790M突变表现出100%的灵敏度和86.96%的特异性。此外，这项工作还完成了血浆中突变等位基因的监测。总之，开发的芯片在肺癌基因分型和治疗监测以实现精准医学方面具有潜力，甚至在其他肿瘤方面也有潜力。
