多靶标病原体单分子核酸扩增分析

Multiplex detection of pathogen provides convenience for screening of various infectious diseases. Obtaining the target information of single nucleic acid molecule is crucial for improving sensitivity and accuracy of clinical diagnosis. Existing multiplex assay methods always suffer from time-consuming and modest sensitivity, thus are not capable to provide information at single-molecule level. In this project, we make a detailed research about single molecule detection and multiplex amplification of nucleic acids as well as controllable fabrication of functional hydrogel, and try to propose a new method for multiplex detection of pathogen nucleic acids based on single molecule amplification. Structure and properties of the crosslinked network in hydrogel should be regulated via changing the monomers and cross linking manners. Then the separated micro-chamber is formed for compartmentalizing single nucleic acid molecules. Molecular recognition and diffusion will be explored at micro/nano scale to increase the efficiency of single molecule amplification, so the multiplex amplification system of single molecule is established. Different pathogen information can be collected once via multi-colour fluorescence imaging. After photobleaching the read signal, new primers are added to initiate the next round of amplification. In this way, we developed a periodic method of multiple single molecule amplification and fluorescence imaging analysis. It improves the quality and quantity of molecular information picked from single assay, and acquires information of multiple pathogen form trace clinical sample timely and accurately, which would provide new ideas for the prevention of related diseases and the monitoring of sudden epidemic.

多病原体同时检测可快速筛查多种疾病，获取靶标核酸单分子信息是提高疾病诊断灵敏度与准确度的关键。现有多检测方法耗时长，灵敏度低，难以提供单分子信息。本项目围绕核酸单分子检测与多重扩增及功能水凝胶可控构筑等问题进行研究，拟构建多靶标病原体单分子核酸扩增分析新方法。筛选预聚合单体、改变交联方式，调控水凝胶网络结构与性能，有效捕获多元长链核酸，形成彼此独立的单分子靶标微反应室；探究微纳限域空间的分子识别与扩散，提高单分子扩增效率，设计系列特异引物探针，建立多靶标核酸单分子多重扩增体系；采集多通道荧光图像，一次性获取多种病原体靶标信息；随后利用光漂白淬灭已读荧光信号并引入新引物探针，对其它待测靶标核酸进行下一轮扩增检测。由此循环，建立周期性的多重单分子扩增与荧光成像分析方法，提升单一样本采集信息的质量与数量，及时、准确获取微量临床样本中的多靶标病原体信息，为相关疾病预防与突发疫情监测提供新思路。

多种病原体同时检测可快速筛查多种疾病，获取靶标核酸单分子信息是提高疾病诊断灵敏度与准确度的关键。现有多检测方法耗时长，灵敏度低，难以提供单分子信息。本项目围绕核酸多重扩增、单分子检测及功能水凝胶可控构筑等问题进行研究，构建了多靶标病原体单分子核酸扩增分析新方法。主要研究内容与重要结果包括：.1、建立基于多基因组合探针与通用指数扩增的多重核酸扩增体系，通过设计并优化特异识别探针与多色荧光探针结构，确保体系多基因高效扩增的同时，显著消除了非特异性扩增引起的背景干扰，可准确识别靶标序列的碱基差异，分辨率可达单个碱基，已成功用于新冠病毒的多基因联合检测与乙肝病毒的基因分型。.2、构建水凝胶核酸单分子扩增微室，通过调控水凝胶孔径尺寸，使其在允许较小分子反应物扩散运动的同时，有效固定长链靶标核酸，建立局部限域的扩增反应，探究微纳尺度的分子识别与扩增效率，为多重扩增反应提供相对独立、平行的单分子微反应区室，实现了多种乙肝病毒的单分子定量与数字化分型。.3、开发了基于核酸组合编码扩增的多色荧光成像策略，通过设计分级DNA支链组装的纳米梯结构探针，精准调控荧光探针的信号强度，提升荧光信号种类。设计大规模环形条码文库，搭建多轮循环组合编码成像平台，建立周期性多循环荧光成像分析方法，实现了150种靶标RNA的多轮编码成像。.本项目实施期间，项目负责人获得国家自然科学基金杰出青年项目资助，荣获2021年度陕西高等学校科学技术一等奖，共培养博士研究生12人，其中4人已于项目在研期间顺利毕业；硕士研究生8人，其中3人已于项目在研期间顺利毕业。相关研究成果已发表SCI论文15篇，获得国家授权发明专利2项，。本项目的完成为快速、准确获取多靶标病原体信息提供了新方法，对推动相关疾病预防与突发疫情监测具有积极推动作用。