A sensitive one-pot ROA assay for rapid miRNA detection

IF 4.6 4区 农林科学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY aBIOTECH Pub Date : 2024-03-18 DOI:10.1007/s42994-024-00140-0
Zhihao Hou, Wenpeng Deng, Alun Li, Ya Zhang, Jianye Chang, Xinyue Guan, Yuxiao Chang, Kaile Wang, Xinjie Wang, Jue Ruan
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Abstract

MicroRNAs (miRNAs) and short RNA fragments (18–25 nt) are crucial biomarkers in biological research and disease diagnostics. However, their accurate and rapid detection remains a challenge, largely due to their low abundance, short length, and sequence similarities. In this study, we report on a highly sensitive, one-step RNA O-circle amplification (ROA) assay for rapid and accurate miRNA detection. The ROA assay commences with the hybridization of a circular probe with the test RNA, followed by a linear rolling circle amplification (RCA) using dUTP. This amplification process is facilitated by U-nick reactions, which lead to an exponential amplification for readout. Under optimized conditions, assays can be completed within an hour, producing an amplification yield up to the microgram level, with a detection limit as low as 0.15 fmol (6 pM). Notably, the ROA assay requires only one step, and the results can be easily read visually, making it user-friendly. This ROA assay has proven effective in detecting various miRNAs and phage ssRNA. Overall, the ROA assay offers a user-friendly, rapid, and accurate solution for miRNA detection.

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用于快速检测 miRNA 的灵敏的一次性 ROA 分析法
微RNA(miRNA)和短RNA片段(18-25 nt)是生物研究和疾病诊断中的重要生物标志物。然而,主要由于其丰度低、长度短和序列相似性,准确快速地检测它们仍然是一项挑战。在这项研究中,我们报告了一种高灵敏度、一步式 RNA O-circle 扩增(ROA)测定法,用于快速准确地检测 miRNA。ROA 检测法首先将圆形探针与检测 RNA 杂交,然后使用 dUTP 进行线性滚圆扩增(RCA)。U-nick 反应促进了这一扩增过程,从而导致指数扩增读数。在优化条件下,检测可在一小时内完成,扩增产率可达微克级,检测限低至 0.15 fmol(6 pM)。值得注意的是,ROA 检测只需一个步骤,而且结果可以很容易地直观读取,方便用户使用。这种 ROA 检测法已被证明能有效检测各种 miRNA 和噬菌体 ssRNA。总之,ROA 检测法为 miRNA 检测提供了一种用户友好、快速而准确的解决方案。
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CiteScore
7.70
自引率
2.80%
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0
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