海产品中病原体的爆发不仅会带来严重的经济损失，更给公众带来巨大的生命威胁。本项目以海产品对虾中两种重要的病原体——副溶血性弧菌和IHHNV为研究对象，开发简便、快速、可视化的双目标同时检测方法。首先，通过可编程的CRISPR/Cas诱导NEAR恒温扩增，将检测信号在短短几分钟内进行百万倍放大；然后利用上转换荧光纳米粒子-分子信标（UCNP-MB）探针和上转换荧光纳米粒子-磁珠（UCNP-MP）探针，通过观察溶液中荧光颜色变化和外加磁场作用下荧光聚集与否对扩增产物进行逻辑判断（AND/OR），实现对双靶标的同时、可视化、高灵敏、特异性检测。该方法集成了上转换荧光纳米粒子信噪比高、分子信标检测特异性强、磁珠运动可控的优势。本项目构建的可视化生物传感器为多目标物的同时、快速、可视化、高灵敏检测提供了广阔前景，可推广应用于农业/食品/公共卫生领域对病原体的快速筛查。

Seafood pathogens usually lead to great economic loss and induce huge threaten to public health. In this project, we plan to develop rapid and sensitive visual detection method for infectious hypodermal and haematopoietic necrosis virus (IHHNV) and vibrio parahaemolyticus detection from shrimp samples at the same time. Firstly, clustered regularly interspersed short palindromic repeats associated (CRISPR/Cas) induced niking enzyme amplification reaction (NEAR) is carried out to make target amplification for million times. Then, with the probe of upconversion nanoparticles and molecular beacon (UCNP-MB), vibrio parahaemolyticus can be detected visually by judging fluorescence change from orange to green in the reaction solution. Simultaneously, with the probe of UCNP and magnetic nanoparticle (UCNP-MP), IHHNV can be detected by observing the aggregation of fluorescence. Through logical judgement of fluorescent color change and fluorescence aggregation (AND/OR), we can detect two targets simultaneously in the same reaction. This method is rapid, sensitive, specific and can be used for multiple targets detection visually at the same time. It is owning to advantages of high signal-to-noise ratio of UCNPs, good paramagnetic effect of magnetic beads and high specificity of molecular beacon. This method shows great prospect for visual detection of multiple targets at the same time, injecting new ideas for rapid and visual pathogen detection in fields of agriculture, food safety and public health.