纸基数字PCR技术的研发及其在核酸检测中的应用

Digital PCR is an emerging absolute quantitative analysis technique, showing great prospects in nucleic acid testing. The key step involved in digital PCR is to achieve single dispersion of nucleic acid molecules. However, existed techniques for sample dispersion are subjected to high cost, poor portability and equipment dependence. Thus we developed a novel nucleic acid dispersion method through combining paper substrate with stretchable PDMS substrate in this program. Here, the sample diffusion and single molecule dispersion can be realized by shrinking or stretching the paper/PDMS substrate. Based on this method, we are devoted to developing a paper-based digital PCR technique. Firstly, the mechanism of sample diffusion in paper porous structure is investigated by simulation analysis; Then, a low-cost, miniaturized and portable paper-based digital PCR platform is developed by integration of the dispersion chip with thermal cycler and image analysis device; Finally, the proposed platform is used to absolute quantitative detection of target nucleic acid. These results can enrich the theoretical mechanism of fluid flow in porous materials and promote the digital PCR technique into point-of-care testing.

数字PCR技术是一种新兴的绝对定量分析技术，在核酸检测领域展现出巨大的应用前景。数字PCR技术的关键是实现核酸分子的单分散。针对现有的数字PCR单分散技术存在成本高、便携性差、依赖仪器使用等问题，本项目拟采用纸基底和可拉伸的PDMS基底相结合的方式，通过收缩/拉伸基底实现核酸样本的自动扩散和单分散。基于此单分散方式，开发一种纸基数字PCR新技术：通过建立微观尺度模拟分析阐明纸基多孔结构内核酸分子的扩散机制与扩增规律；通过集成纸基单分散芯片、热循环装置、图像分析装置，进一步开发低成本、小型化、便携式的纸基数字PCR检测平台；应用开发的纸基数字PCR检测平台实现目标核酸的绝对定量检测。本项目的研究成果可丰富多孔材料内流体流动理论体系，并推动数字PCR技术进入即时检测领域。

以核酸为检测标志物的体外诊断技术得到了大力发展，现已广泛应用于疾病诊断、食品安全等领域。并随着精准医疗和分级诊疗政策的推进，核酸检测领域呈现出单分子精准检测以及低成本、便携式、集成化检测的需求。数字PCR技术在核酸单分子检测需求方面展现出优异的性能，但受限于多台实验室大型仪器，具有成本较高、便携性差、操作复杂等缺点，限制了其更广范围的应用。基于此，本项目构建了耦合宏观毛细流动以及微观孔隙生化反应过程的纸基多孔结构生物分子扩散模型，获得了最优的孔结构参数；在此基础上，开发了纸基核酸提取技术和微流控数字PCR芯片用于核酸的绝对定量分析，并搭建了配套的便携式、集成化的热循环装置和图像读取装置。最终应用开发的数字PCR平台实现了肺癌EGFR基因L858R突变的绝对定量检测。通过与商业数字PCR仪器检测结果进行对比分析，验证了所开发数字PCR检测平台的检测性能。本项目的研究成果对微流控芯片的设计开发以及微流控数字PCR技术的推广应用具有重要意义。