Multicolor and Ultrasensitive Enzyme-Linked Immunosorbent Assay Based on the Fluorescence Hybrid Chain Reaction for Simultaneous Detection of Pathogens

Various pathogens may coexist in one sample; however, detection methods that rely on traditional selective culture media or immune agents designed specifically for a certain target are unsuitable for multiple targets. It is important to develop a simultaneous and sensitive detection method for multiple pathogens. Here, a multicolor and ultrasensitive enzyme-linked immunosorbent assay (ELISA) platform based on the fluorescence hybridization chain reaction (HCR) was developed. In the assay, multicolor fluorescence concatemers formed as signal amplifiers and signal reporters in the presence of target pathogens. When HCR occurred, Escherichia coli O157:H7, Salmonella serotype Choleraesuis, and Listeria monocytogenes were detected simultaneously with three different fluorescences. Additionally, the limits of detection for E. coli O157:H7, Salmonella Choleraesuis, and L. monocytogenes were 3.4 x 10(1), 6.4 x 10(0), and 7.0 x 10(1) CFU/mL, respectively. The assay achieved ultrasensitive, specific, and simultaneous detection of three pathogens and can be applied to the detection of pathogens in milk samples. Therefore, this multicolor and ultrasensitive ELISA platform has great potential in the application of simultaneous detection of pathogens.

一个样本中可能存在多种病原体；然而，依赖传统选择性培养基或针对特定目标专门设计的免疫试剂的检测方法不适用于多个目标。开发一种对多种病原体同时进行灵敏检测的方法至关重要。在此，开发了一种基于荧光杂交链式反应（HCR）的多色超灵敏酶联免疫吸附测定（ELISA）平台。在该测定中，在目标病原体存在的情况下，多色荧光多联体作为信号放大器和信号报告分子形成。当发生HCR时，可同时用三种不同的荧光检测大肠杆菌O157:H7、猪霍乱沙门氏菌和单核细胞增生李斯特氏菌。此外，大肠杆菌O157:H7、猪霍乱沙门氏菌和单核细胞增生李斯特氏菌的检测限分别为3.4×10¹、6.4×10⁰和7.0×10¹菌落形成单位/毫升。该测定实现了对三种病原体的超灵敏、特异和同时检测，并可应用于牛奶样本中病原体的检测。因此，这种多色超灵敏ELISA平台在病原体同时检测的应用中具有很大潜力。