基于微流控-CFPA检测水产品中副溶血性弧菌的新方法研究

副溶血性弧菌（VP）是主要的食源性致病菌之一，目前VP检测方法仍然有诸多不足：检测周期长，操作繁琐，易出现假阳性等。因此，亟需发展简便、快捷、高特异性VP检测新方法以满足日常监控需求。本项目拟结合微流控芯片技术，发展基于结构特异性内切酶FEN1和BstDNA聚合酶的环状荧光探针介导的等温核酸扩增方法（CFPA）检测VP。其主要研究内容包括：1）针对VP中三种溶血素的目标基因（tdh、trh、tlh）和特异性毒力表达调控基因toxR为靶向基因，开发基于微流控-CFPA的高灵敏、高特异性、高通量、多靶点的VP检测新方法，并对其性能进行评价；2）研究重叠结构产生的位置对CFPA方法的影响，对其机理进行补充；3）将该方法应用于海南地区对虾等海产品中VP的检测，分析三种毒力基因表达量在海南分布情况，为VP的日常监控和致病因素溯源提供支持，并为开发便携式VP检测设备提供理论参考。

Vibrio parahaemolyticus (VP) is one of the main food-borne pathogens. Currently, VP detection methods often have disadvantages such as long detection cycle, complicated operation and false positive. Therefore, it is urgent to develop simple, fast and high specificity VP detection methods to satisfy the daily demand.This project intends to develop a highly specific circular fluorescent probe mediated isothermal nucleic acid amplification technique(CFPA) based on FEN1 and Bst DNA polymerase for VP detection by combining with microfluidic chip technology. The main research include the following aspects： (1) Aiming at the target genes of three hemolysins in VP (tdh、trh、tlh) and the specific virulence expression regulation gene (toxR), as target genes, this project constructed a new method for VP detection based on microfluidic CFPA with high sensitivity and throughput and multi-target, which is evaluated about its performance.(2) By changing the position of overlapping structure in the design of probe primers, the influence on CFPA was investigated, and the amplification mechanism was further revealed.(3) This method was applied to detect VP in shrimp from Hainan area, and to clarify the distribution of the expression levels of three virulence genes in shrimp and the difference of hemolytic toxin activity.This method can also be extended to other aquatic products for VP detection, providing a daily monitoring of food-borne pathogens, and the theoretical reference for the development of portable fluorescence detection equipment.