Niu Chenqi, Liu Juewen, Xing Xinhui, Zhang Chong
MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China; Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada.
Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada.
Anal Chim Acta. 2023 Apr 22;1251:340998. doi: 10.1016/j.aca.2023.340998. Epub 2023 Feb 22.
Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3'-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.
非特异性扩增是核酸扩增中的一个主要问题，会导致假阳性结果，尤其是对于指数扩增反应（EXPAR）。尽管人们努力抑制非特异性扩增的影响，如对模板3'端进行化学封闭以及非序列依赖性地减弱模板-模板相互作用，但在许多传统的EXPAR反应中，这仍然是一个常见问题。在本研究中，我们提出了一种新策略，通过将CRISPR-Cas12a系统整合到双模板EXPAR中来消除非特异性扩增产生的非特异性信号。我们开发了一种名为EXPCas的EXPAR-Cas12a策略，其中Cas12a系统充当过滤器，滤除EXPAR中的非特异性扩增子，抑制并消除非特异性扩增的影响。结果，使用该方法信号背景比从1.3提高到了15.4。以微小RNA-21（miRNA-21）为靶标，检测可在40分钟内完成，检测限为103 fM，未观察到非特异性扩增。
