A sensitive one-pot ROA assay for rapid miRNA detection  

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

机构地区：[1]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 [2]Hubei Key Laboratory of Agricultural Bioinformatics,College of Informatics,Huazhong Agricultural University,Wuhan 430070,China [3]Department of Systems Biology,UT MD Anderson Cancer Center,Houston,TX 77030,USA

出　　处：《aBIOTECH》2024年第3期298-308,共11页生物技术通报（英文版）

基　　金：supported the National Key R&D Program of China(2019YFA0707003 and 2022YFC3400300 to J.R.);the Innovation Program of Chinese Academy of Agricultural Sciences.

摘　　要：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.

关 键 词：miRNA Rolling circle amplification(RCA) Sensitive detection Fluorescence detection 

分 类 号：Q522[生物学—生物化学]