The global outbreak of the monkeypox virus (MPXV) highlights the need for rapid and cost‐effective MPXV detection tools to effectively monitor and control the monkeypox disease. Herein, we demonstrated a portable CRISPR–Cas‐based system for naked‐eye detection of MPXV. The system harnesses the high selectivity of CRISPR–Cas12 and the isothermal nucleic acid amplification potential of recombinase polymerase amplification. It can detect both the current circulating MPXV clade and the original clades. We reached a limit of detection (LoD) of 22.4 aM (13.5 copies/µl) using a microtiter plate reader, while the visual LoD of the system is 75 aM (45 copies/µl) in a two‐step assay, which is further reduced to 25 aM (15 copies/µl) in a one‐pot system. We compared our results with quantitative polymerase chain reaction and obtained satisfactory consistency. For clinical application, we demonstrated a sensitive and precise visual detection method with attomolar sensitivity and a sample‐to‐answer time of 35 min.

The outbreak of the monkeypox virus (MPXV) in non-endemic countries is an emerging global health threat and may have an economic impact if proactive actions are not taken. As shown by the COVID-19 pandemic, rapid, accurate, and cost-effective virus detection techniques play a pivotal role in disease diagnosis and control. Considering the sudden multicountry MPXV outbreak, a critical evaluation of the MPXV detection approaches would be a timely addition to the endeavors in progress for MPXV control and prevention. Herein, we evaluate the current MPXV detection methods, discuss their pros and cons, and provide recommended solutions to the problems. We review the traditional and emerging nucleic acid detection approaches, immunodiagnostics, whole-particle detection, and imaging-based MPXV detection techniques. The insights provided in this article will help researchers to develop novel techniques for the diagnosis of MPXV.

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