State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital , Fudan University , Shanghai , 200432 , China.
College of Biosystems Engineering and Food Science , Zhejiang University , Hangzhou , 310058 , China.
Anal Chem. 2019 Oct 1;91(19):12156-12161. doi: 10.1021/acs.analchem.9b01526. Epub 2019 Sep 11.
A rapid and sensitive method is crucial for nucleic acid detection. Recently, RNA-guided CRISPR/Cas12a nuclease-based methods present great promise for nucleic acid detection. In the present methods, however, DNA amplification and subsequent Cas12a cleavage is separated and the whole process takes as long as 2 h. Most importantly, the uncapping operation increases the risk of aerosol contamination. In this study, we propose a CRISPR/Cas12a-based method named "Cas12aVDet" for rapid nucleic acid detection. By integrating recombinase polymerase amplification (RPA) with Cas12a cleavage in a single reaction system, the detection can be accomplished in 30 min and uncapping contamination can be avoided. The detection signal can be observed by the naked eye under blue light. This method could detect DNA at single molecule level and demonstrated 100% accuracy for mycoplasma contamination detection, presenting great potential for a variety of nucleic acid detection applications.
一种快速灵敏的方法对于核酸检测至关重要。最近，基于 RNA 引导的 CRISPR/Cas12a 核酸酶的方法为核酸检测提供了很大的前景。然而，在目前的方法中，DNA 扩增和随后的 Cas12a 切割是分开进行的，整个过程需要长达 2 小时。最重要的是，开盖操作增加了气溶胶污染的风险。在本研究中，我们提出了一种基于 CRISPR/Cas12a 的方法，称为“Cas12aVDet”，用于快速核酸检测。通过将重组酶聚合酶扩增（RPA）与 Cas12a 切割整合在单个反应体系中，检测可以在 30 分钟内完成，并且可以避免开盖污染。检测信号可以在蓝光下通过肉眼观察到。该方法可以在单分子水平上检测 DNA，并在支原体污染检测中表现出 100%的准确性，为各种核酸检测应用提供了巨大的潜力。
