基于CRISPR/Cas识别原理的纸芯片研究及其在肿瘤基因突变、甲基化可视化检测的应用

The rapid development of sequencing technology has prompted the discovery of more biomarkers of tumor genes, which provides a cornerstone for humans to understand the pathogenesis of diseases such as cancer, and for molecular diagnostics and personalized medicine applications. At present, tumor gene detection methods used clinically in China are time-consuming, expensive, and dependent on foreign technology, which consumes a large amount of medical resources and aggravates the burden on patients. Therefore, it is of great significance to develop a portable genetic detection technology with high sensitivity and specificity. Based on the principle of CRISPR recognition, this project aims to construct a visual genetic detection platform for single (test strips) and multiple (paper arrays) detection format by studying sgRNA design, signal probe construction and signal output methods, paper chip design and optimization. Taking the tumor biomarkers EGFR and SEPT9 genes as examples, new methods for detecting gene mutations and gene methylation will be developed. The detection parameters would be evaluated by further application to detection in clinical tumor samples and comparison with the gold standard method. The possibility of non-invasive tumor gene detection would also be evaluated by peripheral blood sampling test. The success of this project is expected to provide an advantage for rapid screening of tumor genes and is expected to be extended to other areas of genetic testing.

测序技术的迅猛发展促使更多的肿瘤基因生物标志物得以发现，这为人类认识癌症等疾病的致病机理，为分子诊断、个性化医学等提供了基石。当前临床使用的肿瘤基因检测方法存在耗时、昂贵、依赖于国外技术等问题，消耗大量医疗资源且加重患者负担。因此发展便携式，具有检测简便性，同时又具备灵敏度高、特异性好等特点的基因检测技术具有重要的意义。本项目基于CRISPR的基因识别原理，通过研究sgRNA设计、信号探针构建及信号输出方法、纸芯片设计与优化等，拟构建单元（试纸条）和多元（纸阵列）的可视化基因检测平台。以肿瘤基因标志物EGFR、SEPT9基因为例，发展新型的基因突变、基因甲基化检测方法。通过进一步应用到临床肿瘤样品中的检测以及与金标准方法的比对，评价其检测参数。通过对外周血采样检测，评价其在无创的肿瘤基因检测中的可能性。本项目的成功有望为肿瘤基因快速筛查提供一种优势的方法，并有望推广到其它基因检测领域。

项目负责人在项目的资助下，三年来带领课题组基于多学科背景，以交叉技术的角度开展生物、医学分子诊断方法和技术的开发和应用研究工作。在 “定性和定量核酸检测新方法”研究以及肿瘤等重要疾病诊断应用上取得了若干进展。包括：1、发明的 Droplet Cas13/Cas12 assay 可实现免核酸扩增的单分子DNA/RNA绝对定量检测，是有优势的新一代定量生物检测平台，是PCR技术发明30多年来核酸检测领域的一个重要检测概念进步；2、开发了低成本、快速的纳米金-核酸探针标记体系，建立了更精确的CASLFA试纸条平台，加速推进自助式家庭核酸测试技术研究进步；3、发明了光控的CRISPR方法，解决了CRISPR检测和核酸扩增的不兼容性难题，提高检测灵敏度并简化操作步骤。研究成果在PNAS, Nat. Comm., J. Am. Chem. Soc., Angew. Chem. Int. Ed., Nano Lett., ACS Nano, Analytical Chemistry等领域重要刊物发表，并受Annu Rev Anal Chem主编邀请撰写该领域综述论文。