快速灵敏的单管检测方法，利用 RPA 耦合 CRISPR-Cas12 检测猴痘病毒。
Rapid and sensitive one-tube detection of mpox virus using RPA-coupled CRISPR-Cas12 assay.
机构信息
NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology and Center for AIDS Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, P.R. China.
NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P.R. China.
出版信息
Cell Rep Methods. 2023 Oct 23;3(10):100620. doi: 10.1016/j.crmeth.2023.100620. Epub 2023 Oct 16.
Mpox is caused by a zoonotic virus belonging to the Orthopoxvirus genus and the Poxviridae family. In this study, we develop a recombinase polymerase amplification (RPA)-coupled CRISPR-Cas12a detection assay for the mpox virus. We design and test a series of CRISPR-derived RNAs(crRNAs) targeting the conserved D6R and E9L genes for orthopoxvirus and the unique N3R and N4R genes for mpox viruses. D6R crRNA-1 exhibits the most robust activity in detecting orthopoxviruses, and N4R crRNA-2 is able to distinguish the mpox virus from other orthopoxviruses. The Cas12a/crRNA assay alone presents a detection limit of 10 copies of viral DNA, whereas coupling RPA increases the detection limit to 1-10 copies. The one-tube RPA-Cas12a assay can, therefore, detect viral DNA as low as 1 copy within 30 min and holds the promise of providing point-of-care detection for mpox viral infection.
猴痘是由一种属于正痘病毒属和痘病毒科的人畜共患病毒引起的。在本研究中，我们开发了一种重组酶聚合酶扩增（RPA）-耦合 CRISPR-Cas12a 检测方法，用于检测猴痘病毒。我们设计并测试了一系列针对正痘病毒保守的 D6R 和 E9L 基因以及猴痘病毒独特的 N3R 和 N4R 基因的 CRISPR 衍生 RNA（crRNA）。D6R crRNA-1 在检测正痘病毒方面表现出最强大的活性，而 N4R crRNA-2 能够将猴痘病毒与其他正痘病毒区分开来。Cas12a/crRNA 检测方法的检测限为 10 个拷贝的病毒 DNA，而耦合 RPA 可将检测限提高到 1-10 个拷贝。因此，一管式 RPA-Cas12a 检测方法可在 30 分钟内检测到低至 1 个拷贝的病毒 DNA，有望为猴痘病毒感染提供即时检测。
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