摘要
为建立一种适用于现场快速检测牛结节性皮肤病(lumpy skin disease,LSD)的方法,将重组酶介导等温扩增(recombinase aided amplification,RAA)技术与侧流层析试纸条(lateral flow dipstick,LFD)相结合,针对牛结节性皮肤病病毒(LSDV)GPCR基因保守区域序列构建质粒,并设计RAA-nfo特异性引物和探针,通过优化RAA-LFD反应体系及条件以及进行特异性、敏感性和重复性评估,成功建立了LSDV RAA-LFD检测方法。该方法在36℃条件下恒温反应25 min,即能实现对LSDV目的基因片段的有效扩增,在LFD显色4 min后可判读结果,且与牛病毒性腹泻病毒、牛诺如病毒、牛冠状病毒、牛轮状病毒等病原无交叉反应,最低检出限为1.15×10^(1) copies/μL,组内重复性试验结果差异不显著(P>0.05)。使用该方法与实时荧光PCR方法对30份模拟临床样品进行检测,二者阳性符合率一致。综上所述,本研究建立的LSDV RAA-LFD检测方法具有操作简便、反应快速、无需精密仪器、对人员要求低等优势,适用于海关等部门开展LSDV现场快速筛查。
In order to rapidly detect lumpy skin disease(LSD)on site,a recombinant plasmid was constructed based on the conserved sequence of GPCR gene of lumpy skin disease virus(LSDV),then using the combination of recombinase aided amplification(RAA)and lateral flow dipstick(LFD),specific RAA-nfo primers and probe were designed accordingly,following optimization of the RAA-LFD reaction system and conditions,as well as comprehensive evaluation of its specificity,sensitivity and reproducibility,a LSDV RAA-LFD assay was successfully established.The assay was capable of effectively amplifying the target LSDV gene fragment under a constant temperature of 36℃ for 25 minutes,and interpreting results after 4 minutes of LFD chromogenic reaction.No crossreactivity was observed with pathogens such as bovine viral diarrhea virus,bovine norovirus,bovine coronavirus or bovine rotavirus.The limit of detection(LOD)was as low as 1.15×10^(1)copies/μL,no significant difference was observed in the intra-group repeatability test(P>0.05).When applied to 30 simulated clinical samples,the positive coincidence rate of this assay was consistent with that of real-time fluorescent PCR.In conclusion,the LSDV RAALFD assay established in the study,with the advantages of accessibility,rapidity,no sophisticated instruments needed,and low skills for operators,was appropriate for on-site rapid screening of LSDV by customs and other quarantine authorities.
作者
徐新龙
吴远扬
程相宁
李子帆
于校杰
马雪连
彭国刚
李兰
Xu Xinlong;Wu Yuanyang;Cheng Xiangning;Li Zifan;Yu Xiaojie;Ma Xuelian;Peng Guogang;Li Lan(Alashankou Customs Technical Center,Alashankou 833418,Xinjiang,China;College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,Xinjiang,China;Key Laboratory of New Drug Research and Development for Herbivorous Animals,Urumqi 830052,Xinjiang,China)
出处
《中国动物检疫》
2026年第4期88-95,共8页
China Animal Health Inspection
基金
海关总署科研项目(2024HK225)
新疆维吾尔自治区重点研发计划项目(2024B02011-2)。
