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A rapid monkeypox virus nucleic acid detection system was developed by integrating recombinase polymerase isothermal amplification(RPA) and clustered regularly interspaced short palindromic repeats-associated protein 12a(CRISPR/Cas12a). The system was combined with lateral flow assay(LFA) to construct an RPA-CRISPR/Cas12a-based rapid nucleic acid detection platform for MPXV, enabling visual and rapid detection. The optimal reaction conditions for RPA were determined to be 37 ℃ for 15 minutes. For the CRISPR system, the optimal volumes of Cas12a, crRNA, NEBuffer, and ssDNA were identified as 1.0, 0.25, 3.0, and 0.25 μL, respectively. The entire detection process was completed within 1 hour, and no cross-reactivity was observed with inactivated influenza A/B virus stock solutions or plasmid controls for vaccinia and variola. The limit of detection(LOD) was confirmed to be 10 copies/μL. A highly sensitive, specific, and rapid RPA-CRISPR/Cas12a-based method for MPXV detection was successfully developed. The method was demonstrated to be applicable to on-site testing and was proven a simple and rapid solution for MPXV detection. By integrating isothermal amplification, CRISPR-based specificity, and LFA visualization, the practicality of this method in resource-limited settings was significantly enhanced. The study highlights the potential of this approach as a promising tool for early outbreak control and public health surveillance.
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Basic Information:
DOI：10.13705/j.issn.1671-6841.2024197
China Classification Code:R511;R440
Citation Information:
[1]DONG Xiaoxu,NIU Nannan,ZHAO Yinzhen ,et al.Establishment of a RPA-CRISPR/Cas12a-based Gene Detection Method for Monkeypox Virus[J].Journal of Zhengzhou University(Natural Science Edition),2026,58(01):87-94.DOI:10.13705/j.issn.1671-6841.2024197.
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河南省科技攻关项目(232102311036)
2024-12-10
2024
2025-02-25
2025
2
2025-12-12
2025-12-12