摘要
本研究建立了一种基于重组酶介导的扩增技术(RAA)以及CRISPR/Cas12a系统快速检测尼帕病毒(NiV)的方法。针对NiV N和F基因分别设计RAA引物和crRNA,构建反应体系,通过前期对引物、crRNA、反应温度以及反应时间等条件筛选优化建立起高效灵敏的检测方法,并评估了该方法的敏感性、特异性和重复性。结果显示:建立的方法在37℃恒温条件下,1h即可完成检测,最低检测限能够达到102拷贝/μL,敏感性较高;特异性强,与口蹄疫病毒(FMDV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV-2)等常见猪病毒均无交叉反应;稳定性较好,对低、中、高浓度病毒质粒均能成功检出且一致性较好。结果表明,本试验建立的NiV快速检测方法操作简单、特异性强、灵敏度高、稳定性好,不需要复杂设备,在蓝光下通过肉眼即可观察结果,可为实现NiV早期快速检测提供有力的技术支持。
A method based on recombinase aided amplification(RAA)and CRISPR/Cas 12a system for rapid detection of Nipah virus(NiV)was established in the study.RAA primers and crRNA were designed respectively based on NiV N and F genes to construct a reaction system,and an efficient and sensitive method was established through screening and optimizing primers,crRNA,temperature and time for reaction and other conditions at early stage,followed by evaluation on its sensitivity,specificity and repeatability.The results showed that the established reaction could be used to complete detection within 1 hour at a constant temperature of 37℃,with the detection limit of 10~2 copies/μL,indicating a high sensitivity;it produced no cro ss-reaction with foot-and-mouth disease virus(FMDV),porcine parvovirus(PPV)and porcine circovirus type 2(PCV-2)due to its strong specificity;and it could detect viral plasmids at low,medium and high concentrations,with good stability and consistency.In conclusion,the established method was with simple operation,strong specificity,high sensitivity and good stability,and the results could be visually observed under blue light without any complex instruments,providing strong technical support for rapid detection of NiV at early stage.
作者
徐蛟
王英丽
王莹
常星
张永强
李金明
包静月
王志亮
Xu Jiao;Wang Yingli;Wang Ying;Chang Xing;Zhang Yongqiang;Li Jinming;Bao Jingyue;Wang Zhiliang(China Animal Health and Epidemiology Center,Qingdao,Shandong 266032,China)
出处
《中国动物检疫》
CAS
2023年第10期95-99,111,共6页
China Animal Health Inspection
基金
“十四五”国家重点研发计划项目(2022YFD1800500)。
