State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Province Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, China.
Hubei Provincial Center for Disease Control and Prevention, Wuhan, 430079, China.
Biosens Bioelectron. 2024 Sep 1;259:116400. doi: 10.1016/j.bios.2024.116400. Epub 2024 May 16.
CRISPR based nucleic acid detection technology provides a deployable approach to point of care testing. While, there remain challenges limiting its practical applications, such as the need for pre-amplification and the long turnaround time. Here, we present a self-cascade signal amplification method with LwaCas13a and an artificially designed "U" rich RNA of stem-loop structure (URH) for pre-amplification-free ultra-fast and ultra-sensitive point-of-care testing (PASSPORT). The PASSPORT system contains: URH, crRNA targeted the URH, crRNA targeted the interesting RNA, fluorescent RNA reporter and LwaCas13a. The assay realized the detection of 100 copies/mL, within 5 min. The PASSPORT platform was further adopted for the detection of marker gene from SASR-CoV-2 and Severe fever with thrombocytopenia syndrome virus (SFTSV), respectively, and 100% accuracy for the analysis of clinical specimens (100 SASR-CoV-2 specimens and 16 SFTSV specimens) was obtained. Integrated with a lateral flow assay device, this assay could provide an alternative platform for the development of point of care testing (POCT) biosensors. PASSPORT has the potential to enable sensitive, specific, user-friendly, rapid, affordable, equipment-free and point-of-care testing for the purpose of large-scale screening and in case of epidemic outbreak.
基于 CRISPR 的核酸检测技术为即时检测提供了一种可部署的方法。然而，其实际应用仍存在一些限制，例如需要预扩增和较长的周转时间。在这里，我们提出了一种无预扩增的自级联信号放大方法，使用 LwaCas13a 和人工设计的茎环结构（URH）富含“U”的 RNA 进行预扩增，用于超快速和超灵敏的即时检测（PASSPORT）。PASSPORT 系统包含：URH、靶向 URH 的 crRNA、靶向感兴趣 RNA 的 crRNA、荧光 RNA 报告分子和 LwaCas13a。该检测方法可在 5 分钟内检测到 100 拷贝/mL 的目标物。该 PASSPORT 平台进一步用于检测 SARS-CoV-2 和发热伴血小板减少综合征病毒（SFTSV）的标记基因，对 100 份 SARS-CoV-2 临床样本和 16 份 SFTSV 临床样本的分析均达到了 100%的准确率。与横向流动检测设备集成后，该检测方法可为即时检测（POCT）生物传感器的开发提供替代平台。PASSPORT 有望实现敏感、特异、用户友好、快速、经济、无设备和即时检测，适用于大规模筛查和疫情爆发的情况。
