Toehold-Containing Three-Way Junction-Initiated Multiple Exponential Amplification and CRISPR/Cas14a Assistant Magnetic Separation Enhanced Visual Detection of Mycobacterium Tuberculosis

作者全名："Chen, Mengqi; Jiang, Xue; Hu, Qianfang; Long, Jinyan; He, Jianwei; Wu, Yuchen; Wu, Zhili; Niu, Yanhong; Jing, Chunmei; Yang, Xiaolan"

作者地址："[Chen, Mengqi; Long, Jinyan; Wu, Yuchen; Wu, Zhili; Niu, Yanhong; Yang, Xiaolan] Chongqing Med Univ, Coll Lab Med, Key Lab Med Lab Diagnost Educ Minist, Chongqing 400016, Peoples R China; [Jiang, Xue] Sichuan Univ, West China Hosp, Dept Lab Med, Chengdu 610041, Sichuan, Peoples R China; [Hu, Qianfang] Chongqing Med Univ, Affiliated Hosp 1, Dept Resp & Crit Care Med, Chongqing 400016, Peoples R China; [He, Jianwei] Army Med Univ, Daping Hosp, Inst Surg Res, Dept Clin Lab Med, Chongqing 400042, Peoples R China; [Jing, Chunmei] Chongqing Med Univ, Childrens Hosp, Chongqing 400015, Peoples R China"

通信作者："Yang, XL (通讯作者)，Chongqing Med Univ, Coll Lab Med, Key Lab Med Lab Diagnost Educ Minist, Chongqing 400016, Peoples R China."

来源：ACS SENSORS

ESI学科分类：CHEMISTRY

WOS号：WOS:001152650700001

JCR分区：Q1

影响因子：8.2

年份：2023

卷号：9

期号：1

开始页：62

结束页：72

文献类型：Article

关键词：three-way junction; multiple exponentialamplification; CRISPR/Cas14a; visual detection; Mycobacteriumtuberculosis

摘要："Rapid and simple nucleic acid detection is significant for disease diagnosis and pathogen screening, especially under specific conditions. However, achieving highly sensitive and specific nucleic acid detection to meet the time and equipment demand remains technologically challenging. In this study, we proposed a magnetic separation enhanced colorimetry biosensor based on a toehold-containing three-way junction (TWJ) induced multiple isothermal exponential amplification and the CRISPR/Cas14a (C-TEC) biosensor. The TWJ template was designed as a Y-X-Y structure. In the presence of the target, the formation of toehold-containing TWJ complex induced primer extension, leading to the generation of amplified single-stranded DNA; this amplified DNA could then bind to either the free TWJ template for EXPAR reaction or the toehold of the TWJ complex for toehold-mediated strand displacement, thereby enabling the recycling of the target. The amplification products could trigger CRISPR/Cas14a for efficient trans-cleavage and release the magnetically bound gold nanoparticle probes for colorimetry detection. Using Mycobacterium tuberculosis 16S rDNA as the target, the proposed C-TEC could detect 16S rDNA down to 50 fM by the naked eye and 20.71 fM by UV-vis detector at 520 nm within 90 min under optimal conditions. We successfully applied this biosensor to clinical isolates of Mycobacterium tuberculosis. In addition, the C-TEC biosensor also showed feasibility for the detection of RNA viruses. In conclusion, the proposed C-TEC is a convenient, fast, and versatile platform for visual detection of pathogen DNA/RNA and has potential clinical applications."

基金机构："National Natural Science Foundation of China [82273900, 82172619]; National Natural Science Foundation of China [20210132]; Science and Technology Planning Project of Yuzhong District of Chongqing City [CSTB2023NSCQ-MSX0994]; Natural Science Foundation of Chongqing"

基金资助正文："This study was supported by the National Natural Science Foundation of China (no. 82273900, 82172619), Science and Technology Planning Project of Yuzhong District of Chongqing City under Grant No. 20210132, Natural Science Foundation of Chongqing (no. CSTB2023NSCQ-MSX0994)."