基于DNAzyme催化介导双循环等温信号扩增策略的高灵敏牛结核杆菌传感方法研究  

A DNAzyme Induced Isothermal Dual-cycle Targets Recycling Amplification for Sensitive Detection of Mycobacterium Bovis

作　　者：傅昕[1] 杨苑 张何[1] 杨梅[1] 张培柔 刘琼 李倩[1] FU Xin;YANG Yuan;ZHANG He;YANG Mei;ZHANG Pei-Rou;LIU Qiong;LI Qian(Hunan Provincial Key Laboratory of Environmental Catalysis and Waste Recycling,College of Materials and Chemical Engineering,Hunan Institute of Engineering,Xiangtan 411104,China)

机构地区：[1]湖南工程学院材料与化工学院,湖南省环境催化与废弃物再生化重点实验室,湘潭411104

出　　处：《分析化学》2022年第12期1822-1831,共10页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.21005067);湖南省重点研发计划项目(No.2020SK3019);湖南省自然科学基金(No.2019JJ40055)资助。

摘　　要：建立了一种8-17 DNAzyme催化介导双循环等温信号放大的牛结核分枝杆菌高灵敏荧光传感方法。牛结核杆菌靶序列与探针H_(1)杂交,导致其发夹结构发生变化,发夹环部缩小,此时Pb^(2+)激活的8-17 DNAzyme活性中心作用于切割位点,释放出引发链和牛结核杆菌靶序列,释放的牛结核杆菌靶序列被重新利用,与H_(1)杂交构建上游循环。引发链与H_(2)杂交打开其发夹结构,随后信号链S_(3) 与打开后的H_(2)结合,并被Pb^(2+)激活的8-17 DNAzyme活性中心识别,切割释放出荧光报告基团,打开的H_(2)可重复利用与S_(3)杂交结合并切割,构建下游循环。在最优实验条件下,对牛结核分枝杆菌特异性序列检测的线性范围为200 amol/L~1 pmol/L,检出限为100 amol/L(S/N=3),回归方程为F=4.834lgC(牛结核杆菌靶序列,amol/L)+4.057。将此方法应用于牛血液中牛结核分支杆菌含量检测,加标回收率在93.2%~107.1%之间。本方法操作简单、选择性好、灵敏度高,检测时间只需60 min,可用于牛血中牛结核分枝杆菌的高灵敏、快速检测。A highly sensitive fluorescent sensing method for Mycobacterium bovis detection by 8-17 DNAzyme mediated dual-cycle isothermal signal amplification was developed.When Mycobacterium bovis target sequence encountered the double loop,it would hybridize with the probe H1,resulting in the change of its hairpin structure and the shrinkage of the hairpin loop,and then the 8-17 DNAzyme active center catalyzed by Pb^(2+) would act on the cleavage site and release the primer strands used for downstream inspiration and the Mycobacterium bovis target sequences which were utilized again in the upstream.As the primer strand hybridized with H_(2),the hairpin structure of H_(2) would gradually open,and then the signal chain S_(3) bound to the opened H_(2) composing a hybrid chain which would be recognized by the 8-17 DNAzyme active center catalyzed by Pb^(2+).Finally,the DNAzyme cleaved and released a fluorescent reporter group,and the opened H_(2) could hybrid with S_(3) and trigger cleavage again.Under the optimal experimental conditions,the linear range for detection of Mycobacterium bovis specific sequences was 200 amol/L-1 pmol/L,with a detection limit of 100 amol/L(S/N=3),and the regression equation was F=4.834lgC(Bovine tuberculosis target sequence,amol/L)+4.057.This method was applied to the detection of Mycobacterium bovis in bovine blood with recoveries of 93.2%-107.1%.The method had the advantages such as simple operation,good selectivity,high sensitivity with a reaction time of 60 min,and it could be used for the highly sensitive and rapid detection of Mycobacterium bovis in bovine blood.

关 键 词：8-17 DNAzyme 双循环信号放大 牛结核分枝杆菌 等温扩增 

分 类 号：S852.618[农业科学—基础兽医学] O657.3[农业科学—兽医学]