CRISPR-Cas12a介导的适配体荧光传感器快速检测金黄色葡萄球菌  

Rapid detection of Staphylococcus aureus by aptamer fluorescence sensor mediated by CRISPR-Cas12a

作　　者：田道明 周子萱 杨迎澳 毛泽峰 任舒悦 韩铁 TIAN Daoming;ZHOU Zixuan;YANG Ying’ao;MAO Zefeng;REN Shuyue;HAN Tie(Military Medical Sciences Academy,Academy of Military Sciences,Tianjin 300050,China)

机构地区：[1]军事科学院军事医学研究院,天津300050

出　　处：《中国医药科学》2024年第21期139-143,173,共6页China Medicine And Pharmacy

基　　金：国家重点研发计划(2021YFC2301104);国家自然科学基金青年项目(82003465)。

摘　　要：目的以金黄色葡萄球菌为典型食源性致病菌代表,利用规律间隔成簇短回文重复序列方法(CRISPR)-相关蛋白(Cas)建立典型食源性致病菌检测新技术。方法在金黄色葡萄球菌存在的情况下,金黄色葡萄球菌可与磁珠上吸附的金黄色葡萄球菌适配体相结合,将原本结合的互补链释放到溶液中,通过磁分离将含有游离的互补链上清液分离出来,与特异性设计的CRISPR RNA(crRNA)结合,激活Cas12a蛋白的反式切割DNA酶活性,对核酸荧光探针进行切割,随后检测体系的荧光强度。结果本方法在前处理完成后,检测反应可控制在1 h内,远低于国标法所需时间,且所需仪器设备简便,易于研发检测试剂盒。与市售金黄色葡萄球菌快速检测试剂盒相比,本实验方法不需要进行前增菌及裂解步骤,耗时较短,且定性结果可满足日常监督检查工作需要。结论本方法操作简便、易于上手、耗时较短,已研制成试剂盒,可与配套便携式检测箱一起使用,方便食堂、驻训等现场对金黄色葡萄球菌进行快速定性筛检。该方法也具有广泛适用性,针对其他食源性致病菌,可通过修改为特定适配体及设计对应crRNA引物,实现不同食源性致病菌的检测。Objective To take Staphylococcus aureus as the representative of typical foodborne pathogens,so as to establish a new detection technology of typical foodborne pathogens by using the method of clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein(Cas).Methods In the presence of Staphylococcus aureus,it could bind to the aptamer of Staphylococcus aureus adapter adsorbed on magnetic beads.The originally bound complementary chains were released into the solution,and the supernatant containing free complementary chains was separated through magnetic separation.Specially designed CRISPR RNA(crRNA)was bound to activate the trans-cleaving DNA enzyme activity of Cas12a protein.the nucleic acid fluorescent probe was cleaved,and then the fluorescence intensity of the system was detected.Results After the pretreatment was completed,the detection reaction could be controlled within 1 hour,which was much shorter than the time required by the national standard method.Moreover,the required instruments and equipment were simple and convenient,and it was easy to develop detection kits.Compared with the commercially available rapid detection kit for Staphylococcus aureus,this experimental method did not need to carry out pre-enrichment and cracking steps,and it took less time,and the qualitative results could meet the needs of daily supervision and inspection work.Conclusion This method is easy to operate,easy to use,and takes a short time.A reagent kit has been developed,and it can be used together with a portable detection box to facilitate rapid qualitative screening of Staphylococcus aureus in canteens,training stations and other fields.This method also has a wide range of applicability.For other food-borne pathogens,different food-borne pathogens can be detected by modifying them into specific aptamers and designing corresponding crRNA primers.

关 键 词：金黄色葡萄球菌 适配体 信号放大 磁性微球 CRISPR-Cas系统 

分 类 号：TS207.4[轻工技术与工程—食品科学]