PDF(Pubmed)
Abstract：
Monkeypox virus (MPXV), the pathogen responsible for the infectious disease monkeypox, causes lesions on the skin, lymphadenopathy, and fever. It has posed a global public health threat since May 2022. Highly sensitive and specific detection of MPXV is crucial for preventing the spread of the disease. Pyrococcus furiosus Argonaute (PfAgo) is an artificial DNA-guided restriction cleavage enzyme programmable with 5\'-phosphorylated ssDNA sequences, which can be developed to specifically detect nucleic acids of pathogens. Here, a PfAgo-based system was established for the detection of MPXV-specific DNA targeting the F3L gene. A short amplicon of 79 bp could be obtained through a fast PCR procedure, which was completed within 45 min. Two 5\'-phosphorylation guide DNAs were designed to guide PfAgo to cleave the amplicon to obtain an 18 bp 5\'-phosphorylation sequence specific to MPXV, not to other orthopoxviruses (cowpox, variola, and vaccinia viruses). The 18 bp sequence guided PfAgo to cleave a designed probe specific to MPXV to emit fluorescence. With optimized conditions for the PfAgo-MPXV system, it could be completed in 60 min for the detection of the extracted MPXV DNA with the limit of detection (LOD) of 1.1 copies/reaction and did not depend on expensive instruments. Successful application of the PfAgo-MPXV system in sensitively detecting MPXV in simulated throat swabs, skin swabs, sera, and wastewater demonstrated the system\'s good performance. The PfAgo platform, with high sensitivity and specificity established here, has the potential to prevent the spread of MPXV.
摘要：
猴痘病毒(MPXV),导致传染性疾病猴痘的病原体，导致皮肤上的病变，淋巴结病，和发烧。自2022年5月以来，它已构成全球公共卫生威胁。MPXV的高灵敏度和特异性检测对于预防疾病的传播至关重要。PyroccusfuriosusArgonaute(PfAgo)是一种人工DNA指导的限制性切割酶，具有5'-磷酸化ssDNA序列，它可以被开发来特异性地检测病原体的核酸。这里,建立了基于PfAgo的系统，用于检测靶向F3L基因的MPXV特异性DNA。通过快速PCR程序可以获得79bp的短扩增子，在45分钟内完成。设计了两个5'-磷酸化指导DNA来指导PfAgo切割扩增子，以获得MPXV特异性的18bp5'-磷酸化序列，不是其他正痘病毒(牛痘，天花,和牛痘病毒）。18bp序列引导PfAgo切割设计的MPXV特异性探针以发射荧光。在PfAgo-MPXV系统的优化条件下，可以在60分钟内完成提取的MPXVDNA的检测，检测限（LOD）为1.1拷贝/反应，并且不依赖于昂贵的仪器。PfAgo-MPXV系统在模拟咽拭子中灵敏检测MPXV中的成功应用，皮肤拭子,sera,和废水证明了系统的良好性能。PfAgo平台,这里建立了高灵敏度和特异性，有可能阻止MPXV的传播。
