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CRISPR-Cas12a enhanced rolling circle amplification method for ultrasensitive miRNA detection
Microchemical Journal ( IF 4.8 ) Pub Date : 2020-11-01 , DOI: 10.1016/j.microc.2020.105239
Gong Zhang , Lei Zhang , Jingtao Tong , Xianxian Zhao , Jianlin Ren

Abstract 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.

中文翻译:

用于超灵敏miRNA检测的CRISPR-Cas12a增强滚环扩增方法

摘要 MicroRNAs (miRNAs)检测具有高特异性和敏感性,受到广泛关注,因为已报道miRNAs在许多疾病的病理发展中起着至关重要的作用,被认为是疾病诊断和预后的潜在生物标志物。我们在此报告了一种通过整合 CRISPR/Cas 系统和滚动循环扩增 (RCA) 技术的优势,在恒温下检测分子外泌体 miRNA 的高度特异性方法。特别是,所提出的策略被证明获得了高灵敏度,这归因于来自 miRNA 挂锁启动的 RCA 和 CRISPR-Cas12a 触发的特异性切割的双重特异性识别。最终,提议的策略显示出 34 的敏感性。
更新日期:2020-11-01
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