State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, 570228, China.
CAS Key Laboratory of Marine Environmental Corrosion and Bio-fouling, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, 266071, China.
Biosens Bioelectron. 2021 Aug 1;185:113262. doi: 10.1016/j.bios.2021.113262. Epub 2021 Apr 23.
Nucleic acid-based diagnosis using CRISPR-Cas associated enzymes is essential for rapid infectious disease diagnosis and treatment strategies during a global pandemic. The obstacle has been blossomed CRIPSR-Cas based tools that can monitor wide range of pathogens in clinical samples with ultralow concentrations. Here, a universal nucleic acid magneto-DNA nanoparticle system was exploited for the detection of pathogenic bacteria, based on the collateral cleavage activity of CRISPR-Cas14a and tag-specific primer extension. In the system, the target nucleic acids were amplificated and be separated from mixtures by streptavidin-coated magnetic bead. The collateral cleavage activity of CRISPR-Cas14a can be activated via the tag sequence on the target product. Consequently, the fluorophore quencher reporter can be activated by CRISPR-Cas14a, leading to the increasing response. The exploited universal bacterial diagnostic can distinguish six different bacteria strains with 1 cfu/mL or 1 aM sensitivity, which may provide new strategies to construct fast, accurate, cost-effective and sensitive diagnostic tools in environments with limited resources.
基于核酸的诊断使用 CRISPR-Cas 相关酶对于在全球大流行期间快速进行传染病诊断和治疗策略至关重要。障碍在于开发出能够以超低浓度在临床样本中监测广泛病原体的基于 CRIPSR-Cas 的工具。在这里，基于 CRISPR-Cas14a 的旁切活性和标签特异性引物延伸，开发了一种通用的核酸磁 DNA 纳米颗粒系统来检测致病菌。在该系统中，通过链霉亲和素包被的磁性珠从混合物中扩增和分离靶核酸。靶产物上的标签序列可以激活 CRISPR-Cas14a 的旁切活性。因此，CRISPR-Cas14a 可以激活荧光团猝灭报告子，从而增加响应。所开发的通用细菌诊断可以区分 6 种不同的细菌菌株，灵敏度为 1 cfu/mL 或 1 aM，这可能为在资源有限的环境中构建快速、准确、具有成本效益和敏感的诊断工具提供新策略。
