In this work, we constructed a manganese ion enhanced CRISPR fluorescence analysis platform based on magnetic separation technology and CRISPR/Cas12a system for the amplification-free detection of SARS-CoV-2 RNA. Firstly, nucleic acid functionalized magnetic beads and dual-function signal probes were used to identify conserved sequences of target RNA at the same time, and then the target was captured and separated by magnetic enrichment. The dual-function signal probe is used to bridge the recognition of the target and the output of the fluorescence signal. And the CRISPR activation sequence on the signal probe is used to initiate the downstream CRISPR/Cas12a fluorescence system to generate fluorescent signals. In this method, we used manganese ions to replace the traditional magnesium ions to assist the CRISPR/Cas12a system, which significantly enhanced the analysis effect. Moreover, we designed a variety of signal probes for different regions of the target RNA to further improve the signal intensity. This method can accurately detect the target RNA within 45 minutes, and can effectively identify the positive cases of COVID-19 infection in clinical throat swab samples, which has great potential for clinical application.

中文翻译：

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基于锰离子增强CRISPR系统和磁富集的病原体核酸免扩增检测方法


在这项工作中，我们构建了基于磁分离技术和 CRISPR/Cas12a 系统的锰离子增强 CRISPR 荧光分析平台，用于 SARS-CoV-2 RNA 的无扩增检测。首先，使用核酸功能化磁珠和双功能信号探针同时鉴定靶 RNA 的保守序列，然后通过磁富集捕获和分离靶标。双功能信号探针用于桥接靶标的识别和荧光信号的输出。而信号探针上的 CRISPR 激活序列用于引发下游 CRISPR/Cas12a 荧光系统产生荧光信号。在该方法中，我们使用锰离子代替传统的镁离子来辅助 CRISPR/Cas12a 系统，从而显着增强了分析效果。此外，我们针对靶 RNA 的不同区域设计了多种信号探针，以进一步提高信号强度。该方法可在 45 分钟内准确检测出目标 RNA，可有效识别临床咽拭子样本中 COVID-19 感染阳性病例，具有很大的临床应用潜力。