Detection of the mpox virus using a robust recombinase-aided amplification-based approach

In 2022, a global outbreak of mpox was anticipated, with several cases reported in non-endemic countries in early May. Given the challenge of distinguishing the mpox virus (MPXV) from other pathogens based solely on symptoms, there is an urgent need for prompt and reliable MPXV detection methods. In this study, we developed assays using recombinase-aided amplification (RAA) to identify MPXV and evaluated their applicability with clinical samples. The assays were designed to detect the <i>N4R</i> gene of MPXV. All assays demonstrated detection limits of 1 copy/µL within the reaction system and exhibited no cross-reactivity with ectromelia or the TianTan strain of vaccinia virus, confirming their high specificity. Our established assay provides results in less than 50 min. Furthermore, we evaluated our assay using clinical samples from laboratory-confirmed mpox patients and demonstrated that the RAA-based assay is valuable for diagnosing MPXV infections in field and clinic settings, especially in areas with limited laboratory resources. Overall, three RAA-based nucleic acid assays for MPXV were established, providing a powerful tool for efficient, rapid, and specific detection of MPXV infection.