基于多重体积微液滴的数字PCR核酸定量检测新方法研究

Digital PCR based quantitative detection of nucleic acids is one of the most important methods to achieve accurate diagnosis of cancer and other major diseases. However, some obstacles such as low detection repeatability and narrow dynamic range remain to be solved in the existing digital PCR technique using an end-point detection method. In this study, we present a multi-volume microdroplets chip based on dynamic pressure control. With a high-density weak fluorescence detection technology by TDI-CCD linear array scanning, we can get real-time fluorescence amplification curves of each droplet, establish a real-time fluorescence model for single nucleic acid molecule amplification, identify non-specific amplification and reduce accidental error. Furthermore, we propose a merging-analysis algorithm for the multi-volume droplets detecting results to maximize the information obtained in each experiment, increase detection repeatability and expand dynamic range significantly. Finally, with a concentration-adaptive experimental method we build an absolute quantitative empirical model to accurately quantify high concentration samples. By all above researches, we hope to set up a digital PCR quantitative detection platform based on multi-volume microdroplets to widen the dynamic range of nucleic acid detection for 3 orders of magnitude with the digital PCR detection repeatability index CV<3%. And a novel theoretical digital PCR quantitative detection method based on multi-volume microdroplets will formed, which will greatly promote the development of digital PCR technology & instrument and expand its applications.

数字PCR核酸定量检测是实现肿瘤等重大疾病精准诊断的重要手段，然而现有数字PCR终点检测实现核酸定量的方法存在重复精度较低、动态范围较窄的问题。本研究提出基于动态压力控制的多重体积微液滴芯片，通过TDI-CCD线阵扫描高密度微弱荧光检测技术，获取每个液滴的实时荧光扩增曲线，建立核酸单分子扩增实时荧光检测模型，降低非特异性扩增等因素引入的偶然误差；利用多重体积微液滴检测结果合并分析算法实现检测信息最大化，提高检测重复精度，拓宽检测动态范围；然后利用浓度自适应样本修正的实验方法得到高浓度样本绝对定量经验模型，解决数字PCR现有技术难以实现高浓度样本精确定量的问题。通过以上研究，建立基于多重体积微液滴的数字PCR核酸定量检测平台，将检测动态范围扩展3个数量级，在全动态范围内实现检测重复精度指标CV<3%，形成基于多重体积微液滴的数字PCR核酸定量检测新方法，促进数字PCR技术、仪器与应用的发展。

数字PCR是实现核酸精准定量的重要技术，对实现肿瘤等重大疾病精准诊断有重大意义。然而，现有数字PCR方案多采用固定体积的PCR试剂体系，且需要繁琐的操作步骤，使得核酸定量结果存在重复精度较低、动态范围较窄等问题。本研究建立了基于多重体积微液滴的核酸单分子扩增实时荧光检测和数据处理新方法；开发了多重体积微液滴生成微流控芯片以及可集成在反应管内大规模微液滴生成装置，充分简化操作过程，并采用0.15nL和0.55nL两种体积的液滴进行数字PCR功能验证，提高核酸定量检测的重复精度并拓宽检测动态范围；搭建了基于CCD成像的大规模单层微液滴检测装置，为建立核酸单分子扩增实时荧光信号模型奠定实验基础。此外，项目团队共发表SCI论文8篇，申请发明专利7项，已获授权4项；项目申请人获第二届全国创新争先奖。