基于NEAR放大及发射光叠加信号分析的高灵敏可视化双食源性病毒检测方法研究

Food-borne Norovirus (NV) and/or Rotavirus (RV) virus infection has been a serious problem threatening human health and thus drawn global concern. The existing methods related have been challenged by the increasing needs in high sensitivity and multiplex in-field visual detection. To address these two problems, we first employ aptamer contained molecular probe instead of conventional antibody to discriminate interested virus particles, and amplify the signals produced promptly via nicking enzyme amplification reaction (NEAR). Next, we proposed a novel strategy to detect multiple targets simultaneously and visually by analyzing emission lights blending signals. In this design, gold-quantum dots complex is employed and functionalized into fluorescent probe targeting virus-specific reporters obtained from NEAR amplification. Once specific reporter is present, the fluorescent signal will be “turn-on”. And if there are multiple targets, a blending of emission lights will be observed. With this, visual multiplex analysis can be achieved in one-pot without uncapping operation. To sum up, this technique is of high sensitivity and multiplex visual analysis capability, providing attractive new ideas for food-borne virus detection. It can also be expanded to target many other food safety related biomolecules to fulfill the needs for food contaminations monitoring and food-borne disease prevention and control.

食源性诺如病毒（NV）和轮状病毒（RV）对人类健康产生的危害已经引起了全球的广泛关注。然而，现有检测方法在灵敏度和现场多重可视化方面仍面临巨大挑战。本课题拟首先利用包含适配体序列的核酸探针代替传统抗体，全面特异地识别病毒分子，并通过Nicking酶恒温扩增反应（NEAR）对识别信号迅速放大，解决灵敏度低的问题；其次，提出利用发射光信号叠加同时分析多目标的可视化检测策略，结合金-量子点荧光能量共振转移（FRET）体系，设计可由信号扩增所得核酸分子特异性开启的荧光探针，达到One-pot无需开盖多重可视化分析的目的。该方法具有灵敏、多重、可视化的特点，为食源性病毒检测提供了新思路；且可被拓展应用于食品安全领域多类目标分子的现场快速分析，为食品污染物监控、食源性疾病预防控制提供了新技术。

本课题建立了双重信号叠加的恒温扩增检测方法，实现更宽可视窗口的多目标分析，通过信号叠加得到接近补色的颜色变化和更大的对比强度，使得肉眼观察更加容易；利用DNA的可编程性质，建立了基于NEAR的分子识别、传感方法，通过理性设计和引入酶切位点的办法，实现了对LAMP扩增产物的快速特异、可视的分析；深入探讨了DNA适配体与金颗粒相互作用的界面理论，分析了研究DNA适配体及金纳米颗粒复合体构建需要考虑的因素，为进一步研究功能性/结构性DNA分子与金纳米颗粒的相互作用奠定了坚实的基础。总结梳理各阶段的相关实验结果，将获得的研究成果发表在国内外重要学术期刊上，相关学术研究论文共计4篇，其中在国际权威SCI期刊上发表相关学术研究论文4篇（影响因子IF>5的3篇，IF>3的4篇），申请了2项国家相关专利，培养硕士研究生3名，其中2名已取得硕士学位。