Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, 100084, PR China.
Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, 100084, PR China.
Biosens Bioelectron. 2024 Jan 1;243:115771. doi: 10.1016/j.bios.2023.115771. Epub 2023 Oct 18.
High-throughput detection of bacteria at low levels is critical in public health, food safety, and first response. Herein, for the first time, we present a platform based on droplet microfluidics coupling with the recombinase aided amplification (RAA)-assisted one-pot clustered regularly interspaced short palindromic repeats together with CRISPR-associated proteins 13a (CRISPR/Cas13a) assay, and droplet encoding strategy for accurate and sensitive determination of nucleic acids from various foodborne pathogens. The workflow takes full advantage of CRISPR/Cas13a signal amplification and droplet confinement effects, which enhances the detection sensitivity and enables end-point quantitation. Meanwhile, by varying the color of droplets, the number of bacteria detected at the same time is greatly improved. It possesses the capability to simultaneously detect seven different types of foodborne pathogens. Notably, the system is also applied to real food samples with satisfactory results. Overall, in view of superiorities in high sensitivity, outstanding selectivity, and large-scale multiplexing, the one-pot CRISPR/Cas13a-based droplet microfluidic system could be expanded and universalized for identifying other bacteria.
在公共卫生、食品安全和紧急响应中，高通量检测低水平的细菌至关重要。在此，我们首次提出了一种基于液滴微流控技术的平台，该平台结合了重组酶辅助扩增（RAA）辅助的一锅簇状规则间隔短回文重复序列与 CRISPR 相关蛋白 13a（CRISPR/Cas13a）检测和液滴编码策略，用于准确和灵敏地测定来自各种食源性致病菌的核酸。该工作流程充分利用了 CRISPR/Cas13a 信号放大和液滴限制效应，从而提高了检测灵敏度并实现了终点定量。同时，通过改变液滴的颜色，可以大大提高同时检测的细菌数量。它具有同时检测七种不同类型食源性致病菌的能力。值得注意的是，该系统还应用于实际食品样本，结果令人满意。总体而言，鉴于其高灵敏度、出色的选择性和大规模的多重检测能力，基于一锅 CRISPR/Cas13a 的液滴微流控系统可以扩展和普及，用于鉴定其他细菌。
