Yu Peihang, Yang Tiantian, Zhang Decai, Xu Lulu, Cheng Xiaoxue, Ding Shijia, Cheng Wei
The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China.
Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
Anal Chim Acta. 2021 May 15;1159:338404. doi: 10.1016/j.aca.2021.338404. Epub 2021 Mar 23.
As one of the crucial factors associated with human life span and cancer progression, telomerase is regarded as an emerging biomarker for cancer diagnosis. Therefore, a facile, rapid and sensitive approach for telomerase activity detection with point-of-care (POC) diagnosis potential is in great demands. Herein, an all-in-one telomerase activity detection assay was established based on the telomere synthesis activated CRISPR-Cas12a system. A telomerase extension reaction generated telomere repeats sequences (TTAGGG), which was recognized by a customized CRISPR-guided RNA (crRNA) simultaneously, and finally activated a typical trans-cleavage based CRISPR-Cas12a detection assay. With the inherent sensitivity of CRISPR-Cas12a, this approach achieved a great linear regression ranging from 100 to 2000 HeLa cells and a limitation of detection down to 26 HeLa cells. Moreover, by using the proposed method, telomerase can be detected in one pot under isothermal condition (37 °C) by a simple and fast workflow (one step within 1 h). Due to its excellent performance, this all-in-one method shows great potential in POC detection of the telomerase activity.
作为与人类寿命和癌症进展相关的关键因素之一，端粒酶被视为一种新兴的癌症诊断生物标志物。因此，迫切需要一种简便、快速且灵敏的端粒酶活性检测方法，具有即时检测（POC）诊断潜力。在此，基于端粒合成激活的CRISPR-Cas12a系统建立了一种一体化端粒酶活性检测方法。端粒酶延伸反应产生端粒重复序列（TTAGGG），其同时被定制的CRISPR引导RNA（crRNA）识别，最终激活基于典型反式切割的CRISPR-Cas12a检测方法。凭借CRISPR-Cas12a固有的灵敏度，该方法在100至2000个HeLa细胞范围内实现了良好的线性回归，检测限低至26个HeLa细胞。此外，通过使用所提出的方法，端粒酶可以在等温条件（37°C）下通过简单快速的工作流程（1小时内一步完成）在一个反应体系中进行检测。由于其优异的性能，这种一体化方法在端粒酶活性的即时检测中显示出巨大潜力。
