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一种用于快速检测微小RNA的灵敏单步滚环扩增检测法。

A sensitive one-pot ROA assay for rapid miRNA detection.

作者信息

Hou Zhihao, Deng Wenpeng, Li Alun, Zhang Ya, Chang Jianye, Guan Xinyue, Chang Yuxiao, Wang Kaile, Wang Xinjie, Ruan Jue

机构信息

Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120 China.

Hubei Key Laboratory of Agricultural Bioinformatics, College of Informatics, Huazhong Agricultural University, Wuhan, 430070 China.

出版信息

aBIOTECH. 2024 Mar 18;5(3):298-308. doi: 10.1007/s42994-024-00140-0. eCollection 2024 Sep.

DOI:10.1007/s42994-024-00140-0
PMID:39279850
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11399362/
Abstract

UNLABELLED

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.

SUPPLEMENTARY INFORMATION

The online version contains supplementary material available at 10.1007/s42994-024-00140-0.

摘要

未标注

微小RNA（miRNA）和短RNA片段（18 - 25个核苷酸）是生物学研究和疾病诊断中的关键生物标志物。然而，由于它们丰度低、长度短以及序列相似性，其准确快速检测仍然是一个挑战。在本研究中，我们报道了一种用于快速准确检测miRNA的高度灵敏的一步法RNA O环扩增（ROA）检测方法。ROA检测从环形探针与待测RNA杂交开始，随后使用dUTP进行线性滚环扩增（RCA）。这种扩增过程由U缺口反应促进，从而实现指数级扩增以便读出结果。在优化条件下，检测可在一小时内完成，扩增产量可达微克水平，检测限低至0.15飞摩尔（6皮摩尔）。值得注意的是，ROA检测仅需一步，结果可轻松通过肉眼读取，使用方便。这种ROA检测已被证明可有效检测各种miRNA和噬菌体单链RNA。总体而言，ROA检测为miRNA检测提供了一种用户友好、快速且准确的解决方案。

补充信息

在线版本包含可在10.1007/s42994 - 024 - 00140 - 0获取的补充材料。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/6aa40ab062cb/42994_2024_140_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/0e7f71cb5171/42994_2024_140_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/acbf9775eb5e/42994_2024_140_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/70581e9746db/42994_2024_140_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/fab905e2c444/42994_2024_140_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/6aa40ab062cb/42994_2024_140_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/0e7f71cb5171/42994_2024_140_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/acbf9775eb5e/42994_2024_140_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/70581e9746db/42994_2024_140_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/fab905e2c444/42994_2024_140_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/967a/11399362/6aa40ab062cb/42994_2024_140_Fig5_HTML.jpg

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