CRISPR/Cas介导核酸扩增结合磁调控可视化FRET信号鉴别食品物种技术研究

Species identification is of extremely importance to guarantee the quality and safety of foodstuffs and prevent the food fraud. Current methods to authenticate the food species are cumbersome, require sophisticated instrument, and have low sensitivity towards multiple targets. This proposal is to develop a rapid and sensitive visual method for identification of Rainbow Trout and Atlantic Salmon in the foodstuffs based on duplex isothermal amplification. The endogenous gene of Rainbow Trout and Atlantic Salmon will be simultaneously amplified by nicking enzyme amplification reaction (NEAR). To circumvent the requirement of nicking enzyme recognition site on the dsDNA, we will employ CRISPR/Cas to produce the ssDNA template for NEAR. A biosensor for detecting the duplex NEAR product will be constructed based on the FRET signal and magnetic separation ability of nanomaterials. The dye-loaded polymeric nanoparticles, which are ultrbright for super-resolution imaging, will provide sequence-specific two-color ratiometric response towards the endogenous gene of Atlantic Salmon. By bridging the polymeric nanoparticles and the magnetic particles, the endogenous gene of Rainbow Trout will be detected by the aggregation of the fluorescent signals in the magnetic field. The method will provide a rapid and sensitive platform for the identification of the Rainbow Trout and Atlantic Salmon species.

食品物种的准确鉴定是保障消费安全、防止食品欺诈的重要技术手段。针对当前食品物种检测技术存在操作时间长、仪器要求高及难以实现多靶标同时高灵敏分析等局限，本项目拟以大西洋鲑鱼和虹鳟的物种鉴别为例，利用CRISPR/Cas技术介导扩增靶基因，并结合聚合物荧光纳米粒子建立高灵敏可视化荧光分析法。首先，本项目将采用CRISPR/Cas技术介导切刻酶恒温扩增反应（NEAR）高效扩增特征内源基因，解决NEAR用于双链DNA扩增灵敏度低的问题；其次，本项目将利用聚合物荧光纳米粒子的强荧光和高FRET效率及磁性纳米粒子（MPs）的磁分离特性，通过靶基因扩增产物调控聚合物纳米粒子的荧光颜色及在磁场中的聚集响应，实现双靶标同时准确可视化区分。该方法具有灵敏、可视化、双信号同时分析的优点，将为食品中多种源成分快速鉴别提供新方法，为防止食品欺诈、保障食品安全提供有力技术支撑。

食品成分的真实性对保障食品安全、预防食品欺诈具有重要意义。三文鱼是世界上经济价值最高的鱼类之一，但与其相关的食品欺诈问题屡见不鲜。当前，针对三文鱼成分的鉴别尚缺乏快速准确的方法。本项目采用核酸扩增与CRISPR/Cas耦联，建立了针对虹鳟和大西洋鲑鱼准确鉴别的可视方法，取得如下进展：（1）研究聚合物纳米粒子的合成方法及影响FRET效率的因素，构建基于聚合物纳米粒子FRET效应的荧光传感器；（2）探究磁调控聚合物纳米粒子运动轨迹及双重信号可视叠加的稳定性及灵敏度，探究基于多维度信号传感的多重输出策略；（3）研究影响切刻内切酶恒温扩增的反应效率和特异性的因素，建立了高灵敏的核酸扩增方法；（4）研究CRISPR/Cas耦联G四链体/ThT的荧光检测策略，构建低成本的荧光免标记可视检测三文鱼成分的方法；（5）探究三文鱼和大西洋鲑鱼的基因序列差异，构建精准可视的CRISPR/Cas检测策略。（6）拓展研究了基于色相变化的免疫层析试纸条半定量可视检测大西洋鲑鱼的原理。通过本项目资助，合计发表SCI论文6篇，申报发明专利5件（授权2件），培养研究生5名，获得中国分析测试协会科学技术奖1项。项目研究成果到达预期目标。