基于80α噬菌体内溶素与SpyCatcher/SpyTag系统的高效金黄色葡萄球菌检测工具设计  

Development of an Efficient Staphylococcus aureus Detection Tool Based on Bacteriophage Endolysin 80αand SpyCatcher/SpyTag System

作　　者：胡江涛 李之琦 赵锋 赵瑾 肖茂桃 李建[2] HU Jiangtao;LI Zhiqi;ZHAO Feng;ZHAO Jin;XIAO Maotao;LI Jian(College of Food and Biological Engineering,Chengdu University,Chengdu 610106,Sichuan,China;College of Preclinical Medicine,Chengdu University,Chengdu 610106,Sichuan,China)

机构地区：[1]成都大学食品与生物工程学院,四川成都610106 [2]成都大学基础医学院,四川成都610106

出　　处：《食品研究与开发》2024年第19期170-176,218,共8页Food Research and Development

摘　　要：金黄色葡萄球菌(Staphylococcus aureus)广泛分布于自然界,是导致食源性疾病的主要病原体之一。为构建一种简便且灵敏的检测金黄色葡萄球菌的方法,该文将80α噬菌体内溶素模块中有着特异性宿主识别和结合作用的催化结构域与细胞结合域模块(Amidase 3⁃CBD)利用SpyCatcher/SpyTag蛋白交联系统有方向性的固定在Ni磁珠表面从而构建免疫磁珠Ni⁃SpyCatcher⁃SpyTag⁃Amidase 3⁃CBD用于捕获金葡金黄色葡萄球菌,再利用等温扩增(re⁃combinase polymerase amplification,RPA)和CRISPR/Cas12a显色切割即可在37℃下2 h内完成检测,并且人工污染牛奶样品中金黄色葡萄球菌的最低检测限为1×102 CFU/mL。该技术不需要专业技术人员或辅助设备,便可实现对金黄色葡萄球菌的高效检测。Staphylococcus aureus ubiquitous in the nature is one of the main pathogens causing foodborne dis⁃eases.A simple and sensitive method for detecting S.aureus was established with the catalytic domain and cell⁃binding domain module(Amidase 3⁃CBD)of the bacteriophage endolysin 80α(with the functions of specific host recognition and binding),the SpyCatcher/SpyTag system,and Ni magnetic beads.In this way,Ni⁃Spy⁃Catcher⁃SpyTag⁃Amidase 3⁃CBD was established for capturing S.aureus.S.aureus was then detected by recom⁃binase polymerase amplification(RPA)and CRISPR/Cas12a chromogenic cleavage at 37℃.The whole detec⁃tion process was completed within 2 h,and the lowest limit of detection for S.aureus in artificially contami⁃nated milk samples was 1×102 CFU/mL.The technology facilitates efficient detection of S.aureus,dispensing with the requirement for specialized technical personnel or auxiliary equipment.

关 键 词：金黄色葡萄球菌 细胞壁结合域 免疫磁分离技术 SpyCatcher/SpyTag系统 重组酶聚合酶扩增(RPA) CRISPR/Cas12a 

分 类 号：TS207.4[轻工技术与工程—食品科学]