基于RPA-CRISPR/Cas12a的猴痘病毒基因检测方法建立

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关键词：猴痘病毒；簇状规则间隔短回文重复序列相关蛋白12a；重组酶聚合酶等温扩增技术；胶体金试纸条

中图分类号：S855.3;Q503 文献标志码：A

DOI：10. 13705/j. issn.1671-6841. 2024197

Abstract：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/Casl2a）． 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°C 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 0.25μL ， respectively. The entire detection process was completed within 1 hour，and no cros-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.

Key words： MPXV ; CRISPR/Cas12a; RPA; colloidal gold test strip

0 引言

猴痘的临床症状类似于天花，如高烧、疲累、头疼、皮疹等，易与其他痘科病毒感染混淆，且有数据表明其传播能力很强[]。（剩余13052字）