DropCRISPR: A LAMP-Cas12a based digital method for ultrasensitive detection of nucleic acid
DropCRISPR: A LAMP-Cas12a based digital method for ultrasensitive detection of nucleic acid
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DOI：
10.1016/j.bios.2022.114377
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发表时间：
2022-05-21
影响因子：
12.6
通讯作者：
deMello, Andrew J.
中科院分区：
文献类型：
--
作者：
Wu, Hui;Cao, Xiaobao;deMello, Andrew J.
自发现以来，CRISPR/Cas系统已广泛应用于核酸生物传感。然而，当代绝大多数平台仅提供核酸的定性检测，未能实现超灵敏的定量检测。在此，我们报道了一个基于数字液滴的平台（DropCRISPR），该平台将环介导的等温扩增（LAMP）与CRISPR/Cas12a相结合，实现了核酸的超灵敏和定量检测。这是通过一种新型的两步微流体系统实现的，该系统将液滴LAMP与能够将所需的CRISPR/Cas12a试剂注入每个液滴的picoinjector相结合。该方法规避了LAMP与CRISPR/Cas12a的温度不兼容，避免了扩增反应与CRISPR检测之间的相互干扰。对含有鼠伤寒沙门菌（St） invA基因的模型质粒实现了超灵敏的检测（fM水平），在纯细菌培养中可实现检测低至102 cfu/mL。此外，我们证明DropCRISPR平台能够检测原料牛奶样品中的St，而无需额外的核酸提取。DropCRISPR的灵敏度和稳健性进一步证明了基于CRISPR/ cas的诊断平台的潜力，特别是当与最先进的微流体架构相结合时。
Since their discovery, CRISPR/Cas systems have been extensively exploited in nucleic acid biosensing. However, the vast majority of contemporary platforms offer only qualitative detection of nucleic acid, and fail to realize ultrasensitive quantitative detection. Herein, we report a digital droplet-based platform (DropCRISPR), which combines loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a to realize ultrasensitive and quantitative detection of nucleic acids. This is achieved through a novel two-step microfluidic system which combines droplet LAMP with a picoinjector capable of injecting the required CRISPR/Cas12a reagents into each droplet. This method circumvents the temperature incompatibilities of LAMP and CRISPR/Cas12a and avoids mutual interference between amplification reaction and CRISPR detection. Ultrasensitive detection (at fM level) was achieved for a model plasmid containing the invA gene of Salmonella typhimurium (St), with detection down to 102 cfu/mL being achieved in pure bacterial culture. Additionally, we demonstrate that the DropCRISPR platform is capable of detecting St in raw milk samples without additional nucleic acid extraction. The sensitivity and robustness of the DropCRISPR further demonstrates the potential of CRISPR/Cas-based diagnostic platforms, particularly when combined with state-of-the-art microfluidic architectures.