Rapid RNA detection through intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction without amplification

作者全名："Zeng, Hongwei; Zhang, Penghui; Jiang, Xue; Duan, Changyuan; Yu, Yang; Wu, Qiaoming; Yang, Xiaolan"

作者地址："[Zeng, Hongwei; Jiang, Xue; Duan, Changyuan; Yu, Yang; Wu, Qiaoming; Yang, Xiaolan] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China; [Zhang, Penghui] Chongqing Med Univ, Childrens Hosp, Clin Lab Ctr, Chongqing Int Sci & Technol Cooperat Ctr Child Dev, Chongqing, Peoples R China"

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

来源：ANALYTICA CHIMICA ACTA

ESI学科分类：CHEMISTRY

WOS号：WOS:000822980500002

JCR分区：Q1

影响因子：6.2

年份：2022

卷号：1217

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：CRISPR-Cas13a cascade cyclic rapid test room temperature respiratory syncytial virus

摘要："The clinical methods to detect RNA viruses and disease-related RNAs suffer from time-consuming processes, high false-positive rates, or limited sensitivity. Here, we propose a strategy for rapid RNA detection through intra-enzyme chain replacement-mediated Cas13a cascade cyclic reaction without target amplification. A hairpin RNA mediator (a cleavage substrate for target-activated Cas13a) and a guiding RNA recognized by the cleavage product through intra-enzyme chain replacement were designed and optimized. Upon the recognition and binding of the target RNA to the Cas13a/CrRNA complex, Cas13a is initially activated to cleave the mediator, and the cleavage products recognize the corresponding Cas13a/CrRNA complex by intra-enzyme chain replacement and initiate the circular cascade of Cas13a cleavage and activation. The accumulated active Cas13a cleaves fluorescent reporter probe for achieving target RNA detection. This ""mix & read "" RNA detection at room temperature was performed in total 30 min. Using miRNA-21 as the target, the changes in fluorescence intensity were linearly correlated to the concentrations from 10 fM to 50 pM with the detection limit of 75 aM, while no significant changes in fluorescence intensity were detected for non-targets. This method applied to the clinical sputum respiratory syncytial virus-positive samples gave results consistent with those from the clinical fluorescence immunoassay. Thus, intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction for detection of RNA viruses in the ""mix & read "" mode at room temperature is rapid, simple, convenient, and efficient for RNA detection and can be adapted to point-of-care testing for high throughput screening of RNA virus infections."

基金机构："National Natural Science Foundation of China [31570862]; Natural Science Foundation Project of Chongqing [CSTC2012JJA0057, CSTC2019jcyj-msxmX0166]"

基金资助正文："This work is supported by National Natural Science Foundation of China (grant no.31570862) , Natural Science Foundation Project of Chongqing (CSTC2012JJA0057, CSTC2019jcyj-msxmX0166) . We give special thanks to Professor Yanli Wang from the Institute of Biophysics, Chinese Academy of Sciences for supply of the pET-28a-LbuCas13a-Sumo-6 ? his expression vectors."