School of Life Science and Technology, Changchun University of Science and Technology, Changchun, 130022, Jilin, China.
Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, Jilin, China.
Anal Bioanal Chem. 2022 Jan;414(2):1073-1080. doi: 10.1007/s00216-021-03733-6. Epub 2021 Oct 25.
In this study, Lba Cas12a (Cpf1) as one of the CRISPR systems from Lachnospiraceae bacterium was coupled with a hybridization chain reaction (HCR) to develop an electrochemical biosensor for detecting the pathogenic bacterium, Salmonella typhimurium. Autonomous cross-opening of functional DNA hairpin structures of HCR yielded polymer double-stranded DNA wires consisting of numerous single-stranded DNAs, which initiated the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave random single-stranded DNA labeling electrochemical tags on the surface of the electrode. It led to a variation in the electron transfer of electrochemical tags. The polymer double-stranded DNA of HCR was immobilized on dynabeads (DBs) via the S. typhimurium aptamer and released from DBs. The established method could selectively and sensitively quantify S. typhimurium in samples with detection limits of 20 CFU/mL. Our study provides a novel insight for exploring universal analytical methods for pathogenic bacteria based on CRISPR-Cas12a coupled with HCR.
在这项研究中，Lba Cas12a（Cpf1）作为来自lachnospiraceae 细菌的 CRISPR 系统之一，与杂交链式反应（HCR）结合，开发了一种用于检测致病菌鼠伤寒沙门氏菌的电化学生物传感器。HCR 的功能 DNA 发夹结构的自主交叉打开产生了由许多单链 DNA 组成的聚合物双链 DNA 线，这引发了 CRISPR-Cas12a 的转切割活性，以不分选地切割电极表面上标记电化学标签的随机单链 DNA。这导致了电化学标签的电子转移发生变化。HCR 的聚合物双链 DNA 通过鼠伤寒沙门氏菌适体固定在 dynabeads (DBs) 上，并从 DBs 上释放。该方法可以选择性和灵敏地定量检测样品中的鼠伤寒沙门氏菌，检测限为 20 CFU/mL。我们的研究为基于 CRISPR-Cas12a 与 HCR 结合的探索通用致病菌分析方法提供了新的见解。
