Highly sensitive and rapid detection of SARS-CoV-2 via a portable CRISPR-Cas13a-based lateral flow assay
Highly sensitive and rapid detection of SARS-CoV-2 via a portable CRISPR-Cas13a-based lateral flow assay
复制标题
通过基于 CRISPR-Cas13a- 的便携式侧流检测对 SARS-CoV-2 进行高灵敏度、快速检测
DOI：
10.1002/jmv.28096
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发表时间：
2022-09-05
影响因子：
12.7
通讯作者：
Song, Hongbin
中科院分区：
文献类型：
--
作者：
Liu, Hongbo;Chang, Shuailei;Song, Hongbin
为了快速识别严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）感染者并控制冠状病毒病（COVID-19）的传播，迫切需要高灵敏度的现场病毒检测方法。为此，开发了基于成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）的分子诊断方法。在此，基于多酶等温快速扩增、CRISPR-Cas 13 a核酸酶和LFA，建立了针对SARS-CoV-2的CRISPR系统介导的侧流检测（LFA）。为了提高检测限（LoD），除了反应体系中各种组分的浓度外，还筛选了crispr RNA、扩增引物和探针。在基于荧光和免疫层析的测定中，CRISPR检测的LoD均提高至0.25拷贝/μ l。为了增强基于CRISPR的LFA方法的质量控制，在侧流条中使用三线条设计检测甘油醛-3-磷酸脱氢酶作为参考。总共使用CRISPR免疫色谱检测技术检测了52份通过逆转录聚合酶链反应（RT-PCR）检测的COVID-19阳性和101份COVID-19阴性临床样本。结果显示100%的一致性，表明我们的方法的有效性相媲美的RT-PCR。总之，这种方法显著提高了CRISPR介导的LFA的灵敏度和可靠性，并为SARS-CoV-2的现场检测提供了重要工具。
To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and control the spread of coronavirus disease (COVID-19), there is an urgent need for highly sensitive on-site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-based molecular diagnostic method was developed for this purpose. Here, a CRISPR system-mediated lateral flow assay (LFA) for SARS-CoV-2 was established based on multienzyme isothermal rapid amplification, CRISPR-Cas13a nuclease, and LFA. To improve the limit of detection (LoD), the crispr RNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The LoD of CRISPR detection was improved to 0.25 copy/mu l in both fluorescence- and immunochromatography-based assays. To enhance the quality control of the CRISPR-based LFA method, glyceraldehyde-3-phosphate dehydrogenase was detected as a reference using a triple-line strip design in a lateral flow strip. In total, 52 COVID-19-positive and 101 COVID-19-negative clinical samples examined by reverse transcription polymerase chain reaction (RT-PCR) were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT-PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR-mediated LFA and provides a crucial tool for on-site detection of SARS-CoV-2.