High Specific and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based Exponential Rolling Circle Amplification
High Specific and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based Exponential Rolling Circle Amplification
复制标题
基于挂锁探针的指数滚环扩增对 MicroRNA 进行高特异性和超灵敏等温检测
DOI：
10.1021/ac401715k
复制
发表时间：
2013-08-20
影响因子：
7.4
通讯作者：
Tang, Bo
中科院分区：
文献类型：
--
作者：
Liu, Haiyun;Li, Lu;Tang, Bo
本文建立了一种基于挂锁探针的指数滚环扩增（P-ERCA）方法，用于microRNA（miRNA）的高特异性、高灵敏度检测。挂锁探针由与miRNA的杂交序列和用于切口核酸内切酶的切口位点组成。使用miRNA作为模板，在等温条件下实现与挂锁探针的特异性连接和线性滚环反应（LRCA）。在多次切口反应后，连续产生许多拷贝的短DNA产物，然后用作下一个循环扩增的触发物。因此，少量的miRNA被转化为大量的触发物以启动滚环扩增反应，并且实现循环指数信号扩增。该基于挂锁探针的指数滚环扩增测定使用挂锁探针的优化序列表现出0.24 zmol的显著灵敏度。锁式探针的靶点依赖性环化和连接反应可有效提高特异性，实现miRNA家族成员间单核苷酸差异的区分。利用该方法对人肺细胞中的miRNA进行了分析。结果表明，这种高灵敏度的P-ERCA策略将成为一种有前途的miRNA定量方法在早期临床诊断。
In this paper, a padlock probe-based exponential rolling circle amplification (P-ERCA) assay is developed for highly specific and sensitive detection of microRNA (miRNA). The padlock probe is composed of a hybridization sequence to miRNA and a nicking site for nicking endonuclease. Using the miRNA as a template, specific ligation to the padlock probe and linear rolling circle reaction (LRCA) are achieved under isothermal conditions. After multiple nicking reactions, many copies of short DNA products are successively produced and then used as triggers in next circle amplification. Thus, a small amount of miRNAs are converted to a large number of triggers to initiate the rolling circle amplification reaction, and circular exponential signal amplification is achieved. This padlock probe-based exponential rolling circle amplification assay exhibits a remarkable sensitivity of 0.24 zmol using optimized sequences of the padlock probe. The target-dependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to single nucleotide difference discrimination between miRNA family members. The miRNA analysis in human lung cells was performed with this method. The result indicates this highly sensitive P-ERCA strategy will become a promising miRNA quantification method in early clinical diagnostics.