CRISPR-Cas12a enhanced rolling circle amplification method for ultrasensitive miRNA detection
CRISPR-Cas12a enhanced rolling circle amplification method for ultrasensitive miRNA detection
复制标题
CRISPR-Cas12a增强滚环扩增方法用于超灵敏miRNA检测
DOI：
10.1016/j.microc.2020.105239
复制
发表时间：
2020-11-01
影响因子：
4.8
通讯作者：
Ren, Jianlin
中科院分区：
文献类型：
--
作者：
Zhang, Gong;Zhang, Lei;Ren, Jianlin
microRNAs（miRNAs）在多种疾病的发生、发展过程中起着重要作用，被认为是疾病诊断和预后的潜在生物标志物，因此miRNAs的检测具有高度的特异性和敏感性，受到广泛关注。本文结合CRISPR/Cas系统和滚环扩增（rolling circular amplification，RCA）技术的优点，建立了一种高特异性的外泌体miRNAs恒温检测方法。特别地，所提出的策略被证明获得归因于来自miRNA-挂锁引发的RCA和CRISPR-Cas 12 a-触发的特异性切割的双重特异性识别的高灵敏度。最终，所提出的策略显示出34.7 fM的灵敏度，这对于exosomal miRNA的检测是足够稳健的，并且与逆转录定量聚合酶链反应（RT-qPCR）获得了高度的一致性，揭示了开发用于多种疾病的筛查、诊断和预后预测的通用分子检测平台的潜力。
MicroRNAs (miRNAs) detection with high specificity and sensitivity received abundant attention because miRNAs have been reported to play a vital role in pathological development of many diseases and regarded as potential biomarkers for the diagnosis and prognosis of diseases. We reported here a highly specific method for molecular exosomal miRNAs detection in constant temperature by integrating the advantages of CRISPR/Cas system and rolling circular amplification (RCA) techniques. Especially, the proposed strategy was demonstrated to obtain a high sensitivity attributed to the dual-specific recognition from miRNA-padlock initiated RCA and CRISPR-Cas12a-triggered specific cleavage. Eventually, the proposed strategy showed a sensitivity of 34.7 fM which was robust enough for exosomal miRNA detection and obtained a high consistency with reverse transcription quantitative polymerase chain reaction (RT-qPCR), revealing the potential of developing a universal molecular detection platform for the screening, diagnosing, and prognosis prediction of multiple diseases.