posted on 2020-01-09, 14:03authored byRuixuan Wang, Xianxian Zhao, Xiaohui Chen, Xiaopei Qiu, Guangchao Qing, Hong Zhang, Liangliang Zhang, Xiaolin Hu, Zhuoqi He, Daidi Zhong, Ying Wang, Yang Luo
Multiplexed
detection of extracellular vesicle (EV)-derived microRNAs
(miRNAs) plays a critical role in facilitating disease diagnosis and
prognosis evaluation. Herein, we developed a highly specific nucleic
acid detection platform for simultaneous quantification of several
EV-derived miRNAs in constant temperature by integrating the advantages
of a clustered regularly interspaced short palindromic repeats/CRISPR
associated nucleases (CRISPR/Cas) system and rolling circular amplification
(RCA) techniques. Particularly, the proposed approach demonstrated
single-base resolution attributed to the dual-specific recognition
from both padlock probe-mediated ligation and protospacer adjacent
motif (PAM)-triggered cleavage. The high consistency between the proposed
approach RCA-assisted CRISPR/Cas9 cleavage (RACE) and reverse transcription
quantitative polymerase chain reaction (RT-qPCR) in detecting EV-derived
miRNAs’ abundance from both cultured cancer cells and clinical
lung cancer patients validated its robustness, revealing its potentials
in the screening, diagnosis, and prognosis of various diseases. In
summary, RACE is a powerful tool for multiplexed, specific detection
of nucleic acids in point-of-care diagnostics and field-deployable
analysis.