创伤弧菌快速检测法的建立

Development of Rapid Detection of Vibrio vulnificus

[目的]针对创伤弧菌(Vibrio vulnificus)采用杂交瘤技术制备单克隆抗体,基于单克隆斑点杂交技术进行改良,建立一种创伤弧菌快速检测方法。[方法]由水产品牡蛎中分离获得一株创伤弧菌FD-1,以其灭活全菌为抗原免疫小鼠,采用杂交瘤技术制备单克隆抗体。在此基础上,对传统斑点杂交技术进行优化改进,以化学发光试剂ECL替代传统TMB底物,进一步提高其灵敏度、缩短检测时间和简化操作步骤。[结果]与传统斑点杂交法相比,改良斑点杂交法的检测时间可缩短至2 h,检测限可达1×10~5CFU/m L。[结论]改良斑点杂交法比传统TMB底物斑点杂交法(1×10~7CFU/m L)灵敏度提高了100倍,操作过程简单,在水产品检验和医疗诊断方面具有良好的开发应用前景。

[Objective]An ECL-based dot blot assay was developed for the rapid and sensitive detection of Vibrio vulnificus based on MAbs against V. vulnificus.[Method] The FD-1 was isolated from the oyster,taking inactivated whole bacteria as antigen,monoclonal antibody was prepared by hybridoma technique,on this basis,the traditional dot hybridization technology was optimized and improved. The enhanced chemiluminescent（ ECL） substrate was used to replace the traditional TMB substrate,which can further improve the sensitivity,shorten the detection time and simplify the operation steps. [Result]Compared with the traditional dot blot assay,the incubation time was shortened to 2 h. The detection limit can reach 1×10~5 CFU/m L.[Conclusion]The LOD is 100-fold more sensitive than the traditional dot blot assay（ 1×10~7 CFU/m L）. The operation process is simple,and has a good prospect for development and application in aquatic product inspection and medical diagnosis.