摘要
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。
Objective 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.
作者
蔡何清
张旭亮
陈晓娟
汪洌
戴方伟
李巍
CAI Heqing;ZHANG Xuliang;CHEN Xiaojuan;WANG Lie;DAI Fangwei;LI Wei(Laboratory Animal Center of Zhejiang University,Hangzhou 310018,China;Hangzhou Medical College,Hangzhou 310059,China)
出处
《实验动物科学》
2025年第3期23-29,共7页
Laboratory Animal Science
基金
浙江省基础公益研究计划(LGD21C040007)。
作者简介
蔡何清(1996-),女,硕士,研究方向为实验动物检测方法研究,E-mail:16651436946@163.com;通信作者:戴方伟(1978-),女,副研究员,研究方向为实验动物监测及病原诊断方法研究,E-mail:fangweidai@163.com;通信作者:李巍(1980-),女,高级实验师,研究方向为实验动物质量控制,E-mail:liwei0016@zju.edu.cn。
