Wei Jihua, Song Zichun, Cui Jiuying, Gong Yuanxun, Tang Qianli, Zhang Kai, Song Xinlei, Liao Xianjiu
Guangxi Key Laboratory of Basic and Translational Research of Bone and Joint Degenerative Disease, The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi 533000, China.
West Guangxi Key Laboratory for Prevention and Treatment of High-incidence Diseases, Youjiang Medical University for Nationalities, Baise, Guangxi 533000, China.
J Hazard Mater. 2023 Jun 15;452:131268. doi: 10.1016/j.jhazmat.2023.131268. Epub 2023 Mar 22.
In this study, we introduce an electrochemiluminescence (ECL) sensing platform based on the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). This platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The TiCT-compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability. To obtain the T7 promoter, the SARS-CoV-2 RdRp gene may first initiate an entropy-driven cyclic amplification response. Then, after recognizing the T7 promoter sequence on the newly created dsDNA, T7 RNA polymerase starts transcription, resulting in the production of many single-stranded RNAs (ssRNAs), which in turn trigger the action of CRISPR/Cas13a. Finally, Cas13a/crRNA identifies the transcribed ssRNA. When it cleaves the ssRNA, many DNA reporter probes carrying -U-U- are cleaved on the electrode surface, increasing the ECL signal and allowing for the rapid and highly sensitive detection of SARS-CoV-2. With a detection limit of 7.39 aM, our method enables us to locate the SARS-CoV-2 RdRp gene in clinical samples. The detection method also demonstrates excellent repeatability and stability. The SARS-CoV-2 RdRp gene was discovered using the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). The developed ECL test had excellent recoveries in pharyngeal swabs and environmental samples. It is anticipated to offer an early clinical diagnosis of SARS-CoV-2 and further control the spread of the pandemic.
在本研究中，我们介绍了一种基于“熵驱动触发T7扩增 - CRISPR/Cas13a系统”（EDT - Cas）的电化学发光（ECL）传感平台。该平台结合了可编程的熵驱动循环策略、T7 RNA聚合酶和CRISPR/Cas13a系统，用于扩增对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）RNA依赖性RNA聚合酶（RdRp）基因的检测。符合TiCT的ECL信号分子与ECL传感平台一起使用时具有独特优势，可提高检测灵敏度和电极表面可修饰性。为了获得T7启动子，SARS-CoV-2 RdRp基因可能首先引发熵驱动的循环扩增反应。然后，在识别新生成的双链DNA上的T7启动子序列后，T7 RNA聚合酶开始转录，产生许多单链RNA（ssRNA），进而触发CRISPR/Cas13a的作用。最后，Cas13a/crRNA识别转录的ssRNA。当它切割ssRNA时，许多携带 -U-U- 的DNA报告探针在电极表面被切割，增加了ECL信号，从而实现对SARS-CoV-2的快速、高灵敏度检测。我们的方法检测限为7.39 aM，能够在临床样本中定位SARS-CoV-2 RdRp基因。该检测方法还具有出色的重复性和稳定性。利用“熵驱动触发T7扩增 - CRISPR/Cas13a系统”（EDT - Cas）发现了SARS-CoV-2 RdRp基因。所开发的ECL检测方法在咽拭子和环境样本中具有出色的回收率。预计它将为SARS-CoV-2提供早期临床诊断，并进一步控制疫情传播。
