食源性致病菌超灵敏可视化检测：引物探针型SmartAmp扩增协同多位点荧光编码纳米探针技术的研究

Currently, various methods for food-borne pathogens detection have been well-established. However, these methods are still suffering from sophisticated instruments, tedious procedures and relatively low sensitivity. It would be advantageous to develop new methods that are rapid, highly sensitive and specific, along with simple sampling processes and inexpensive instrumentations. Therefore, such methods can be adapted by the laboratories in remote areas for routine food-borne pathogen detections. To satisfy those requirements, we herein propose a novel detection strategy for food-borne pathogens which combines prime-probe SmartAmp and multi-locus fluorescence-encoded nanoprobing technology. For proof-of-concept, we propose to use Salmonella, intestinal hemorrhagic E. coli O157：H7 and Listeria as models to establish and demonstrate a novel ultrasensitive detection strategy for multiplexed detection of food-borne pathogens with the naked eyes. The proposed strategy demonstrates several advantages over the traditional methods. First of all, the SmartAmp amplification can be performed after simple sampling process. Second, the amplification process is equivalent to detection, namely the "absolute specific" signal of the amplification technology. Third, through the introduction of fluorescence-encoded nanoprobing technology, rapid multiplexed detection with the naked eyes can be achieved. No expensive instruments will be required, since water bath for amplification and UV lamp for visual detection are enough for the proposed strategy. Therefore, we believe that this method has the potential to be used as a routine method in the laboratories of remote areas for high-throughput food-borne pathogens detection.

食源性致病菌的检测手段多种多样，但在易操作、灵敏度等方面仍存在不足：如需贵重仪器、操作步骤繁琐、灵敏度低等。能否无需繁琐的样品前处理，即能实现超灵敏简易检测，且不需要贵重仪器，能够在基层实验室推广应用？针对上述问题，本课题选取常见食源性致病菌如肠出血性大肠杆菌O157:H7、沙门氏菌、单增李斯特菌等为研究对象，首次提出引物标记荧光介导的等温扩增（引物探针型SmartAmp）协同多位点荧光编码纳米探针技术，建立一种超灵敏“可视化”同步检测多种食源性致病菌的技术平台。其流程是对样本简单处理后进行SmartAmp扩增，因引物带有标记荧光及其SmartAmp独有的 “绝对特异性”扩增，使其扩增过程即为检测结果，可直接裸眼观察；引入多位点荧光编码纳米探针技术进一步增加其检测通量与灵敏度。因等温扩增和可视化检测无需贵重仪器，常规实验室的水浴锅和紫外灯即可满足，非常适合在基层实验室开展致病菌的筛查工作。

该项目聚焦实际工作中的需求，以食品中的危害因子如食源性致病菌为研究对象发展超灵敏检测技术，把酶循环信号放大策略引入到检测体系，构建多种致病菌的同步可视化超灵敏检测，建立基于引物探针技术、信号循环放大、纳米荧光编码技术等相结合实现目标危害物质的超灵敏可视化检测。.第一部分：首先基于大肠杆菌O157:H7 rfbE 基因、沙门氏菌invA基因、单增李斯特菌inlA设计捕获探针和生物素化的引物序列，结合信号放大技术，构建引物探针型食源性致病菌可视化检测体系。捕获探针固定在载体上，通过与PCR产物进行杂交后，把待检测的目的片段DNA杂交到芯片上。HRP标记的链霉亲和素和生物素化的抗HRP抗体引入到载体平台，形成基于HRP的酶循环信号放大系统，最终通过TMB和H202显色实现三种致病菌的同步超灵敏可视化检测。.第二部分：新型荧光编码纳米探针的制备：通过由“核”到“壳”采用layer-by-layer 层层包裹的共价交联的方式，研制出“核、壳”均能放大荧光信号的超灵敏荧光纳米探针，而不同于目前的荧光纳米发光模式，对该纳米探针可根据包含物质的比例进行调节不同的颜色，实现荧光编码的目标。.第三部分：干燥型荧光PCR扩增技术和等温扩增快速检测食源性致病菌体系的构建：针对食品中常见的致病菌设计PCR引物、SmartAmp引物及探针，构建食源性致病菌的超灵敏可视化同步检测技术平台。采用干燥型荧光PCR技术对三种食源性致病菌的灵敏度、特异性和人工污染食品样品的检测情况进行研究，用高、中、低三个浓度目标菌液人工污染食品样品，按国标培养后用进行对比研究分析。初步建立了基于等温扩增和荧光编码纳米探针相结合的超灵敏可视化检测技术平台。.目前，在项目执行期间共发表论文8篇，其中4篇SCI,4篇科技核心期刊，另外尚有2篇SCI准备投稿中。已申请国家发明专利5项，已获得授权发明专利2项，新型实用专利2项。项目本单位与中山大学联合培养硕士研究生2名，博士1名，项目组成员1人晋升正高，2人晋升副高，参与了2次相关的学术会议交流。