To establish a rapid MHV monitoring method based on environmental media for early pathogen warning in laboratory animal barrier environments. Methods Conserved sequences of MHV were analyzed to design specific RAA primers and crRNA compatible with the LwCas13a reaction system. After optimization, a RAA-CRISPR / Cas13a detection system for MHV was developed, with validation of its specificity and sensitivity, followed by application in animal environmental dust sample detection. Results A rapid MHV detection method based on Recombinase aided amplification ( RAA) combined with CRISPR / Cas13a technology was established for dust samples from laboratory animal habitats. Specificity tests demonstrated no cross-reactivity with common murine viruses including Sendai  virus, Reovirus type 3, Pneumonia virus of mice, Encephalomyelitis virus, and Murine norovirus. The method showed high specificity for MHV detection with a superior sensitivity of 10 copies/ μL detection limit. Clinical validation using PCR as reference involved parallel testing of 160 random dust samples, revealing complete consistency between both methods and identifying 26 antigen-positive samples.Conclusion The RAA-CRISPR / Cas13a detection system based on environmental dust provides efficient and sensitive technical support for pathogen surveillance in laboratory animal facilities.

目的 建立基于环境介质的小鼠肝炎病毒( MHV) 快速监测方法,实现实验动物屏障环境的病原早期预警。方法 分析 MHV 的保守序列,对特异重组酶介导等温扩增(RAA)引物及 CRISPR / Cas13a 反应的 crRNA 进行设计和优化,建立用于检测 MHV 的 RAA-CRISPR / Cas13a 检测体系,并验证其特异性、敏感性。 为进一步验证其实用性,将该检测体系应用于动物环境粉尘样本的检测。 结果针对实验动物生存环境的粉尘样本,建立了基于重组酶介导等温扩增(RAA)结合 CRISPR / Cas13a 技术的 MHV 快速检测方法。 特异性实验结果表明,该方法对仙台病毒( SeV) 、呼肠孤病毒 3 型(Reo-3) 、小鼠肺炎病毒( PVM) 、小鼠脑脊髓炎病毒( MEV) 及小鼠诺如病毒( MNV) 等常见小鼠病毒均无交叉反应;对 MHV 检测具有高度特异性,最低检测限可达 10 copies/ μL,具有优越的检测灵敏度。采用 PCR 法为对照进行临床样本验证,160 份随机粉尘样本进行平行检测,两种方法检测结果完全一致,共检出 26份核酸阳性样本。 结论 基于环境粉尘的 RAA-CRISPR / Cas13a 检测体系为实验动物设施病原监测提供了新的思路及高效灵敏的技术支持。

实验动物, 病原预警方法, 快速检测, CRISPR / Cas13a, MHV