环介导等温扩增
多路复用
多重位移放大
条形码
小RNA
核酸
计算生物学
假阳性悖论
生物
生物系统
分子生物学
DNA
聚合酶链反应
计算机科学
遗传学
基因
DNA提取
操作系统
机器学习
作者
Yuqian Tan,Li Zhang,Shixiong Deng
摘要
Multiple analysis of miRNAs is essential for the early diagnosis and monitoring of diseases. Here, a programmable, multiplex, and sensitive approach was developed for one-pot detection of miRNAs by melting temperature encoded sequences and exponential isothermal amplification (E-EXPAR). In the presence of target miRNAs, the corresponding templates initiate the cycles of nicking and polymerization/displacement, generating numerous barcode strands with unique encoding sequences. Subsequently, generated barcode strands hybridize with fluorescent probes and quench the fluorophore by a triplet of G base through a photo-induced electron transfer mechanism. Finally, a melting curve analysis is performed to quantify miRNAs by calculating the rate of fluorescence change at the corresponding melting temperature. Based on this, miRNA-21, miRNA-9, and miRNA-122 were detected with the detection limits of 3.3 fM, 2.9 fM, and 1.7 fM, respectively. This E-EXPAR was also employed to simultaneously detect three miRNAs in biological samples, showing consistent results with RT-qPCR. Overall, this study provides a programmable and universal platform for multiplex analysis of miRNAs, and holds great promise as an alternative to the multiplex analysis in clinical diagnostics and prognostics for nucleic acid detection.
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