CAS Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430207, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Viruses. 2024 Feb 29;16(3):382. doi: 10.3390/v16030382.
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. Argonaute (Ago) 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 Ago-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 Ago 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 Ago to cleave a designed probe specific to MPXV to emit fluorescence. With optimized conditions for the Ago-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 Ago-MPXV system in sensitively detecting MPXV in simulated throat swabs, skin swabs, sera, and wastewater demonstrated the system's good performance. The Ago platform, with high sensitivity and specificity established here, has the potential to prevent the spread of MPXV.
猴痘病毒（MPXV）是引起猴痘传染病的病原体，可导致皮肤损伤、淋巴结病和发热。自 2022 年 5 月以来，它已构成全球公共卫生威胁。高度敏感和特异的 MPXV 检测对于预防疾病传播至关重要。Argonaute（AGO）是一种人工 DNA 引导的限制酶，可通过 5'-磷酸化的单链 DNA 序列编程，可用于特异性检测病原体的核酸。在这里，建立了一种基于 AGO 的系统，用于检测针对 F3L 基因的 MPXV 特异性 DNA。通过快速 PCR 程序可获得 79bp 的短扩增子，整个过程在 45 分钟内完成。设计了两个 5'-磷酸化的向导 DNA 来引导 AGO 切割扩增子，以获得 18bp 的 5'-磷酸化序列，该序列特异性针对 MPXV，而不针对其他正痘病毒（牛痘、天花和痘苗病毒）。18bp 序列引导 AGO 切割针对 MPXV 的设计探针以发出荧光。在优化的 AGO-MPXV 系统条件下，可在 60 分钟内完成提取的 MPXV DNA 的检测，检测限（LOD）为 1.1 个拷贝/反应，且不依赖于昂贵的仪器。AGO-MPXV 系统在模拟咽喉拭子、皮肤拭子、血清和废水中敏感检测 MPXV 的成功应用表明了该系统的良好性能。在此建立的高灵敏度和特异性的 AGO 平台有可能预防 MPXV 的传播。
