基于“磁三核酸-肽核酸”的食源性致病微生物条形码纸基光学传感器的研究

Food microorganism pollution is a significant problem in the field of food safety. Firstly, in order to challenge the culture-free, highly specific aptamer library will be established, which is going to cover 10 categories of common foodborne pathogens. Secondly, according to the conformation transition function of aptamer, we will explore some important laws, including the composition, association and dissociation of magnetic DNA triplex, specific magnetic enrichment of microorganisms and the signal transformation. As a result, the problems of low specificity and stability of microbial immune enrichment will be solved. Thirdly, nicking endonuclease enzyme-mediated X-Y-efficiency index isothermal amplification technology will be established which can realize the end quantitative and improve the detection sensitivity. Finally, both single and multiple barcode polyamide nucleic acid and paper-based optical sensor will be developed by effective combinations of the above-described attractive theories and techniques, so that we can solve the problem of accurate detection and high throughout screening. This project proposed the theory of “Magnetic DNA Triplex- Peptide Nucleic Acid”, which will solve a series of scientific problems in microbiological detection skillfully, including identification, capture, enrichment, amplification and identification. This will be an ultra-sensitive, practical and efficient solution to screen and identify foodborne pathogenic microorganisms in a fast, accurate and high-throughout pattern.

微生物污染一直都是食品安全中的一个突出问题。为突破微生物免培养检测的难题，针对常见的10类食源性致病菌建立高特异性的核酸适配体库；为解决微生物免疫富集特异性低、稳定性差的问题，依据适配体的构象转变功能提出磁三核酸体概念，探索其构成、结合和解离规律，为微生物的特异性磁富集及信号转导提供理论支撑；为解决免培养检测灵敏度低及等温扩增难终点定量的难题，提出等温指数扩增理论，结合磁三核酸体建立切克内切酶介导的X-Y型等温指数扩增技术，指数放大原始微生物比例的差异，实现终点定量，提高检测灵敏度；为解决现场精准检测及高通量筛查难题，最终建立超灵敏定量检测的单重肽核酸纸基光学传感器及用于高通量筛查检测的条形码纸基光学传感器。本项目提出“磁三核酸-肽核酸”理论，巧妙的解决了微生物从识别、捕获、富集、扩增、现场检测的一系列科学难题，为食源性致病菌的快速、准确、高通量筛查和鉴定提供超灵敏、实用、高效的解决方案。

本项目从微生物污染一直都是食品安全中的一个突出问题出发。为突破微生物免培养检测的难题， 针对常见的10类食源性致病菌建立高特异性的核酸适配体库；为解决微生物免疫富集特异性低、稳定性差的问题，依据适配体的构象转变功能搭建了三核酸分子开关，探索其构成、结合和解离规律及最优工作条件，为微生物的特异性富集及信号转导提供理论支撑；为解决免培养检测灵敏度低及等温扩增难终点定量的难题，提出等温指数扩增理论，结合三核酸分子开关建立切克内切酶介导的X-Y型等温指数扩增技术，指数放大原始微生物比例的差异，实现终点定量，提高检测灵敏度；为解决现场精准检测及高通量筛查难题，最终建立超灵敏定量检测的单重肽核酸纸基光学传感器及用于高通量筛查检测的条形码纸基光学传感器。.本项目研究内容主要分为“病原微生物适配体库”、“三核酸分子开关”、“指数等温扩增反应”、“肽核酸纸基传感器”及“条形码纸基光学传感器”的搭建五大部分，分别建立包含110条适配体的功能核酸适配体库，pH响应及核酸序列可调控的三核酸分子开关，具有高扩增效率的等温指数扩增反应及单重肽核酸纸基传感器和多重条形码纸基传感器。在完成核定任务的基础上，着重进行了等温扩增技术的拓展研究，包括LAMP、HCR、TDT酶介导无模板扩增、RPA、多重连接反应MLPA及PCR反应等温化等反应的研究，并总结功能核酸理论。为食品安全风险因子的现场可视化奠定了强有力的技术支撑。.本项目中通过将核心任务分解，巧妙的解决了微生物从识别、捕获、富集、扩增、现场检测的一系列科学难题，为食源性致病菌的快速、准确、高通量筛查和鉴定提供超灵敏、实用、高效的解决方案。