College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China.
Anal Chem. 2021 Nov 16;93(45):15216-15223. doi: 10.1021/acs.analchem.1c04133. Epub 2021 Nov 4.
The development of a sensitive, facile, and cost-effective colorimetric method is of great significance for the point-of-care testing of viral nucleic acid. Herein, we reported a strand displacement amplification assisted CRISPR-Cas12a (SDACC) method for the colorimetric analysis of viral nucleic acid. The hepatitis B virus (HBV) DNA was chosen as the target to trigger strand displacement amplification (SDA) and generate abundant single-strand DNA (ssDNA) products. The ssDNA amplicon hybridized with template DNA to activate the trans-cleavage activity of CRISPR-Cas12a, leading to the nonspecific cleavage of ssDNA on GOx-ssDNA-modified magnetic beads and the release of GOx. The released GOx was capable of catalyzing the substrate solution to generate a color change, which could be directly observed by naked eyes. The SDACC strategy could identify a single-base mismatch located in the DNA sequence and achieve a sensitive detection for HBV DNA with the limit of detection as low as 41.8 fM. Notably, the sophisticated primer design for target amplification and complicated detection process could be circumvented. The current approach realizes a simple, low-cost, and sensitive colorimetric detection for viral nucleic acid and holds great promise for the practical application of virus infection diagnosis.
开发一种灵敏、简便且经济有效的比色法对于即时检测病毒核酸具有重要意义。在此,我们报道了一种基于链置换扩增辅助 CRISPR-Cas12a(SDACC)的比色法用于病毒核酸的分析。选择乙型肝炎病毒(HBV)DNA 作为靶标来触发链置换扩增(SDA)并产生丰富的单链 DNA(ssDNA)产物。ssDNA 扩增子与模板 DNA 杂交以激活 CRISPR-Cas12a 的转切割活性,导致 ssDNA 在 GOx-ssDNA 修饰的磁性珠上的非特异性切割和 GOx 的释放。释放的 GOx 能够催化底物溶液产生颜色变化,可通过肉眼直接观察到。SDACC 策略能够识别 DNA 序列中单个碱基的错配,并实现对 HBV DNA 的灵敏检测,检测限低至 41.8 fM。值得注意的是,该方法无需复杂的引物设计和繁琐的检测过程。目前的方法实现了病毒核酸的简单、低成本和灵敏的比色检测,为病毒感染诊断的实际应用提供了广阔的前景。