Yan Xiao-Mei, Wang Yin-Qi, Chen Yao, Chen Zeng-Ping, Yu Ru-Qin
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan, 410082, PR China.
Hunan Key Lab of Biomedical Materials and Devices, College of Life Sciences and Chemistry, Hunan University of Technology, Zhuzhou, 412008, PR China.
Anal Chim Acta. 2020 Sep 22;1131:1-8. doi: 10.1016/j.aca.2020.07.025. Epub 2020 Jul 28.
A simple but effective method for the detection of miRNAs was proposed by integrating exonuclease-III assisted target recycling amplification and repeated-fishing strategy. In the proposed method, exonuclease-III assisted target recycling amplification reaction is adopted to produce a large amount of DNA fragments with fluorescence group at its 5' end in the presence of the target miRNA, which are then repeatedly fished out from the reaction mixture by a gold foil modified with a capture probe and transferred into a so-called 'product tube'. The amount of the target miRNA can then be determined from the fluorescence measurement of the solution in the 'product tube'. Application to the detection of miRNA-155 in samples of KH-2 and BRSA-2B cells revealed that the proposed method could achieve sensitive and accurate quantification of the target miRNA with a limit of detection of 36 fM and recovery rates in the range from 96.2% to 105%. Its simplicity, sensitivity and resistance to possible fluorescence interferences in complex biological samples make the proposed method a potentially competitive alternative for miRNAs detection in complex biological samples.
通过整合核酸外切酶III辅助的靶标循环扩增和重复捕捞策略，提出了一种简单而有效的miRNA检测方法。在所提出的方法中，采用核酸外切酶III辅助的靶标循环扩增反应，在靶标miRNA存在的情况下，产生大量5'端带有荧光基团的DNA片段，然后通过用捕获探针修饰的金箔从反应混合物中反复捞出，并转移到所谓的“产物管”中。然后可以通过对“产物管”中溶液的荧光测量来确定靶标miRNA的量。应用于KH-2和BRSA-2B细胞样品中miRNA-155的检测表明，所提出的方法能够实现对靶标miRNA的灵敏和准确定量，检测限为36 fM，回收率在96.2%至105%之间。其简单性、灵敏度以及对复杂生物样品中可能的荧光干扰的抗性，使得所提出的方法成为复杂生物样品中miRNA检测的潜在竞争性替代方法。
