基于CRISPR-Cas12a系统反式切割活性的凝血酶检测技术研究

Study on thrombin detection technology based on trans-cleavage activity of CRISPR-Cas12a system

摘要:

目的 通过构建快速、灵敏的凝血酶检测技术，为多种血管栓塞性疾病，如脑静脉窦血栓形成（cerebral venous sinus thrombosis，CVST）的早期诊断和治疗提供新的策略。方法 利用所筛选的高特异性凝血酶适配体设计发夹结构变构探针，用于高特异性识别凝血酶并将凝血酶信号转化为核酸信号。在变构探针识别凝血酶后，适配体结合凝血酶引发变构探针变构，暴露Cas12a活性部分。CRISPR-Cas12a系统识别活性部分后，其反式切割特性被触发，开始无序切割周围存在的单链DNA荧光探针，导致标记在荧光探针两端的荧光基团（Cy3）和淬灭基团（BHQ）分离，使处于被淬灭状态的Cy3荧光重新出现。所产生的荧光信号的强度与体系中存在的凝血酶浓度呈正相关。结果 通过荧光实验证实所设计的变构探针对凝血酶表现出极高的识别特异性和稳定性；在所优化的实验条件下，该方法表现出良好的检测性能，检测限为0.23 pmol/L。结论 该方法结合了高灵敏度、低成本和良好的便携性等优点，为凝血酶相关疾病的检测与精准诊断提供了强有力的技术支持。

Abstract:

Objective To provide new strategies for the early diagnosis and treatment of a variety of vascular embolic diseases，like cerebral venous sinus thrombosis（CVST），by constructing a rapid and sensitive thrombin detection technology.Methods The screened aptamer，which could recognize thrombin with high specificity，was used to construct the allosteric probe and to convert the thrombin signal into a nucleic acid signal. After the allosteric probe recognized thrombin，aptamer section in allosteric probe bound to thrombin，exposing the Cas12a active moiety. CRISPR-Cas12a system subsequently recognized the active moiety，and then activated its trans-cleavage property to cleave the surrounding single-stranded DNA fluorescent probes disorderly，resulting in separation of a fluorescent group（Cy3） and a quenching group（BHQ） which were originally labeled at both ends of the fluorescent probe. As a result，fluorescence signal of Cy3 reappeared. The intensity of the generated fluorescent signal was positively correlated with the concentration of thrombin present in the system.Results The fluorescence experiment confirmed that the designed allosteric probe showed high recognitive specificity and stability for thrombin. This method exhibited good detection performance in detection，with a limit of 0.23 pmol/L.Conclusion This method combines the advantages of high sensitivity，low cost and good portability，and can provide strong support for the detection and accurate diagnosis of thrombin-related diseases.