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CRISPR/Cas12a技术与重组酶聚合酶扩增技术相结合用于快速便携式检测发热伴血小板减少综合征病毒

CRISPR/Cas12a Technology Combined With RPA for Rapid and Portable SFTSV Detection.

作者信息

Huang Mengqian, Liu Sihua, Xu Yanan, Li Aqian, Wu Wei, Liang Mifang, Niu Guoyu, Wang Zhiyun, Wang Tao

机构信息

School of Life Sciences, Tianjin University, Tianjin, China.

National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.

出版信息

Front Microbiol. 2022 Jan 25;13:754995. doi: 10.3389/fmicb.2022.754995. eCollection 2022.

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a new tick-borne pathogen that can cause severe hemorrhagic fever. Fever with thrombocytopenia syndrome caused by SFTSV is a new infectious disease that has posed a great threat to public health. Therefore, a fast, sensitive, low-cost, and field-deployable detection method for diagnosing SFTSV is essential for virus surveillance and control. In this study, we developed a rapid, highly sensitive, instrument-flexible SFTSV detection method that utilizes recombinase polymerase amplification and the CRISPR/Cas12a system. We found that three copies of the L gene from the SFTSV genome per reaction were enough to ensure stable detection within 40 min. The assay clearly showed no cross-reactivity with other RNA viruses. Additionally, our method demonstrated 100% agreement with Q-PCR detection results for SFTSV in 46 clinical samples. We simplified the requirements for on-site detection instruments by combining the CRISPR/Cas12a tool and immunochromatographic strips to create a system that can reliably detect one copy/μl sample of the L gene, which showed extremely high sensitivity and specificity for detecting the virus. Taken together, these findings indicate that the new SFTSV detection method is a powerful and effective tool for on-site detection, which can contribute to diagnosing SFTSV quickly and sensitively.

摘要

发热伴血小板减少综合征病毒(SFTSV)是一种新型蜱传病原体,可引起严重出血热。由SFTSV引起的发热伴血小板减少综合征是一种对公众健康构成重大威胁的新型传染病。因此,一种快速、灵敏、低成本且可现场部署的SFTSV诊断检测方法对于病毒监测和控制至关重要。在本研究中,我们开发了一种利用重组酶聚合酶扩增和CRISPR/Cas12a系统的快速、高度灵敏且仪器灵活的SFTSV检测方法。我们发现,每个反应中来自SFTSV基因组的L基因三份拷贝足以确保在40分钟内进行稳定检测。该检测方法与其他RNA病毒无交叉反应。此外,我们的方法在46份临床样本中与SFTSV的Q-PCR检测结果一致性达100%。通过将CRISPR/Cas12a工具与免疫层析试纸条相结合,我们简化了现场检测仪器的要求,创建了一个能够可靠检测L基因1拷贝/μl样本的系统,该系统对病毒检测具有极高的灵敏度和特异性。综上所述,这些发现表明新的SFTSV检测方法是一种用于现场检测的强大而有效的工具,有助于快速、灵敏地诊断SFTSV。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/56c3/8822122/59ee8dfe5a65/fmicb-13-754995-g001.jpg

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