Sensitive and specific CRISPR-Cas12a assisted nanopore with RPA for Monkeypox detection.
Sensitive and specific CRISPR-Cas12a assisted nanopore with RPA for Monkeypox detection.
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DOI：
10.1016/j.bios.2023.115866
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发表时间：
2023-11
影响因子：
12.6
通讯作者：
Md. Ahasan Ahamed;Muhammad Asad Ullah Khalid;Ming Dong;Anthony J Politza;Zhikun Zhang;Aneesh Kshirsagar;Tianyi Liu;W. Guan
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文献类型：
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作者：
Md. Ahasan Ahamed;Muhammad Asad Ullah Khalid;Ming Dong;Anthony J Politza;Zhikun Zhang;Aneesh Kshirsagar;Tianyi Liu;W. Guan
猴痘病毒（MPXV）构成全球卫生紧急情况，需要快速、简单和准确的检测，以有效管理其传播。聚类规则间隔短回文重复序列（CRISPR）技术已成为一种有前途的下一代分子诊断方法。在这里，我们开发了一种具有等温重组酶聚合酶扩增（RPA）的高灵敏度和特异性CRISPR-Cas12a辅助纳米孔（SCAN）用于MPXV检测。RPA-SCAN方法提供了非放大扫描无法实现的灵敏度，同时也解决了PCR-SCAN在护理点应用中的障碍。我们证明了单分子的大小计数能够分析裂解报告基因的反应时间依赖性分布。我们的mpxv特异性RPA测定在50 μL反应体系中达到了19个拷贝的检测限（LoD）。通过将2 μL的RPA扩增整合到20 μL的CRISPR反应中，我们使用SCAN传感器获得了16个拷贝/μL （26.56 aM）的MPXV的总LoD，置信水平为95%。我们还验证了RPA-SCAN区分MPXV和牛痘病毒的特异性，准确率为100%。这些发现表明，等温RPA-SCAN装置非常适合于高灵敏度和特异性的猴痘检测。鉴于其电子特性和小型化潜力，RPA-SCAN系统为在护理点诊断广泛的其他感染性病原体铺平了道路。
Monkeypox virus (MPXV) poses a global health emergency, necessitating rapid, simple, and accurate detection to manage its spread effectively. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique has emerged as a promising next-generation molecular diagnostic approach. Here, we developed a highly sensitive and specific CRISPR-Cas12a assisted nanopore (SCAN) with isothermal recombinase polymerase amplification (RPA) for MPXV detection. The RPA-SCAN method offers a sensitivity unachievable with unamplified SCAN while also addressing the obstacles of PCR-SCAN for point-of-care applications. We demonstrated that size-counting of single molecules enables analysis of reaction-time dependent distribution of the cleaved reporter. Our MPXV-specific RPA assay achieved a limit of detection (LoD) of 19 copies in a 50 μL reaction system. By integrating 2 μL of RPA amplifications into a 20 μL CRISPR reaction, we attained an overall LoD of 16 copies/μL (26.56 aM) of MPXV at a 95% confidence level using the SCAN sensor. We also verified the specificity of RPA-SCAN in distinguishing MPXV from cowpox virus with 100% accuracy. These findings suggest that the isothermal RPA-SCAN device is well-suited for highly sensitive and specific Monkeypox detection. Given its electronic nature and miniaturization potential, the RPA-SCAN system paves the way for diagnosing a wide array of other infectious pathogens at the point of care.