基于HCR高灵敏多通量检测食源性致病菌的新型ELISA

Food safety is an important public health priority. Foodborne diseases are serious global problems and impose a significant burden not only on infected individuals but also on the economy. Conventional detection methods require 5 d to 7 d to identify target bacteria. As such, rapid and sensitive detection methods should be developed to screen pathogens in food. Enzyme-linked immunosorbent assay (ELISA) have been extensively investigated. While pre-enrichment, which cost time, is required in the conventional ELISA, slowing the response of state and local food safety department. And the traditional ELISA usually detect single species of bacteria, while a method of simultaneous detecting multiple bacteria is needed for practical application. In this project, A novel ELISA for the sensitive and simultaneous determination of Foodborne Pathogens is built, by using DNA-based hybridization chain reaction (HCR), biotin–streptavidin signal amplification, and ELISA array. Three kinds of polyclonal antibody(pAb) are immobilized in the ELISA wells. The initiator strand (DNA1) and three kinds of monoclonal antibody (mAb) are labeled on gold nanoparticle (AuNP) to form a mAbs–AuNP–DNA1 complex. In the presence of the target, the sandwiched immunocomplex, which is pAb–target–mAb–AuNP–DNA1, could be formed. Two types of biotinylated hairpin were subsequently added in the ELISA well. A nicked double-stranded DNA that contained abundant biotins was formed after HCR. Detection was performed after adding horseradish peroxidase–streptavidin and substrate/chromogen solution. This project offers a promising idea for the sensitive and multiple detection of foodborne pathogens.

食源性致病菌严重危害公共安全，同时导致巨大的经济损失。传统的培养法需要5-7天才能确证目标菌，因此，亟需快速灵敏的检测食源性致病菌方法。酶联免疫吸附法（ELISA）已广泛应用。常规的ELISA需要长时间的预增菌，且一般以单种菌作为目标物，而在实际检测中需要对多种菌进行快速检测。本项目拟集成基于DNA杂交链式反应、生物素-链霉亲和素系统和ELISA阵列技术，建立一种针对食源性致病菌检测的新型高灵敏多通量ELISA方法。三种多抗分别预包被于ELISA孔中，引发链DNA1与三种相应的单抗标记于纳米金上。当目标物存在时，经双抗夹心将形成多抗-目标物-单抗-金-DNA1复合物。之后引入两种生物素标记的发卡型单链DNA，HCR过程后，富含大量生物素的双链DNA在纳米金的表面形成。最后加入链霉亲和素标记的HRP和酶的底物进行信号输出。本项目的实施将为丰富高灵敏多通量的食源性致病菌检测方法提供新思路。

食源性致病菌严重危害公共安全，同时导致巨大的经济损失。传统的培养法需要5-7天才能确证目标菌，常规的ELISA需要烦琐的预增菌，且一般以单种菌作为目标物。本项目集成DNA杂交链式反应、生物素-链霉亲和素系统和ELISA阵列技术，建立了针对食源性致病菌检测的新型高灵敏多通量ELISA方法。为了实现高灵敏检测，我们设计了一种基于静态淬灭(CQ)和荧光共振能量转移(FRET)的低荧光背景发卡(CQ-FRET发卡)。基于CQ和FRET，非特异性信号明显减少。基于CQ-FRET发卡所建立的检测大肠杆菌O157:H7的免疫-HCR方法(2.2×101CFU/mL)比基于CQ发卡(2.45×103CFU/mL)和基于FRET发卡(4.85×104CFU/mL)所建立的免疫-HCR方法更灵敏。为了实现多重检测，我们设计了三套杂交序列，每套的引发链（initiator）仅能引发匹配的H1和H2，并且每套序列的H1上分别标记了互不干扰的荧光基团。ELISA与HCR相结合，存在目标菌时，形成免疫夹心复合物，加入修饰了荧光基团的发夹对，发生HCR，形成多色的荧光级联体作为信号放大分子和输出分子。结果显示，该平台能够实现对E. coli O157: H7、S. Choleraesuis和L. monocytogenes的同时检测，LOD分别为3.4×101、6.4×100和7.0×101CFU/mL，具有良好的特异性，并且该平台也可应用于加标牛奶样本中多重致病菌的检测。本项目的实施将为丰富高灵敏多通量的食源性致病菌检测方法提供新思路。