Amplified Tandem Spinach-Based Aptamer Transcription Enables Low Background miRNA Detection
Amplified Tandem Spinach-Based Aptamer Transcription Enables Low Background miRNA Detection
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DOI：
10.1021/acs.analchem.8b02471
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发表时间：
2018-08-21
影响因子：
7.4
通讯作者：
Li, Jinghong
中科院分区：
文献类型：
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作者：
Tang, Xin;Deng, Ruijie;Li, Jinghong
MicroRNAs(MiRNAs)在调节基因表达和细胞功能方面起着关键作用，被认为是许多人类疾病的潜在生物标志物。临床诊断和治疗对miRNA的检测提出了更高的要求。在这里，我们描述了一种无标记、低背景的荧光分析方法，称为基于菠菜的放大串联适体转录分析(AmptSpi Test)，用于通过聚合物滚动环扩增子介导的多重转录来检测高灵敏的miRNA。目标miRNA被挂锁探针识别，形成聚合的滚环扩增片段。随后的转录过程迅速产生大量重复的RNA菠菜适配子，通过加入荧光染料DFHBI进行miRNA定量分析，实现了无标记和几乎为零的本底。此外，该方法还可以高选择性地区分具有1个或2个核苷酸(NT)差异的miRNA家族成员。该方法能在数小时内完成对人血清样品或细胞提取液的检测，在疾病的早期诊断方面具有很大的潜力。
MicroRNAs (miRNAs) play key roles in regulating gene expression and cell functions, which are recognized as potential biomarkers for many human diseases. Sensitive, specific, and reliable detection of miRNA is highly demanded for clinical diagnosis and therapy. Herein, we describe a label-free and low-background fluorescent assay, termed amplified tandem Spinach-based aptamer transcription assay (AmptSpi assay) for highly sensitive miRNA detection by polymeric rolling circle amplicon mediated multiple transcription. Target miRNA is recognized by padlock probe to form polymeric rolling circle amplicon. Then the following transcription process rapidly produces large amounts of repeats of RNA Spinach aptamers, lightened up by the addition of fluorescent dye DFHBI for miRNA quantitative analysis, achieving label-free and nearly zero-background. Besides, the assay could also confer high selectivity to distinguish miRNA among the miRNA family members with 1- or 2-nucleotide (nt) difference. This method was capable of completing detection in human serum sample or cell extracts in hours, indicating great potential in the early diagnosis of diseases.