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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引用次数: 0

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。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

aBIOTECH

CiteScore

7.70

自引率

2.80%

发文量