锦鲤疱疹病毒RAA-LFD检测方法的建立  

Development of a recombinase-aid amplification assay combined with lateral flow dipstick for detection of the Koi herpesvirus

作　　者：吕晓楠 徐立蒲 张文 王小亮 王静波 王姝 曹欢 LV Xiao-Nan;XU Li-pu;ZHANG Wen;WANG Xiao-liang;WANG Jing-bo;WANG Shu;CAO Huan(Beijing Aquaculture Technology Extention Station,Beijing 100176,China)

机构地区：[1]北京市水产技术推广站,北京100176

出　　处：《中国预防兽医学报》2024年第4期374-378,425,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：现代农业产业技术体系北京市渔业创新团队(BAIC07-2023-10)。

摘　　要：为建立一种简便、快捷的现地检测锦鲤疱疹病毒(KHV)的方法,本研究结合重组酶介导扩增(RAA)和侧向流试纸条(LFD)技术,根据RAA引物设计原则,以KHV Sph基因保守区为靶位点,设计5对引物并筛选出5号引物作为最优引物,在其下游引物、探针的5'端分别标记Biotin、FAM荧光基因。采用方阵法对RAA反应时间和温度等条件优化,结果显示,该检测方法在34℃~40℃恒温反应10 min即可实现对KHV目的基因片段的有效扩增,并且扩增产物结果可直接观察,由此初步建立了KHV的RAA-LFD检测方法。特异性试验结果显示,建立的RAA-LFD检测方法与鲤浮肿病毒(CEV)、鲫造血器官坏死病毒(CHNV)、流行性造血器官坏死病毒(EHNV)、蛙虹彩病毒(BIV)等常见水生动物病原核酸均无交叉反应。敏感性试验结果显示,该方法对重组质粒标准品p EASY-KHV的检测限为50拷贝/μL,与行业标准常规PCR的敏感性相当。重复性试验结果显示,该方法对5份p EASY-KHV的检测结果均一致;对室温放置7 d的p EASY-KHV检测结果也均一致。采用该方法和常规PCR对60份临床样品检测,结果显示二者的阴性、阳性符合率以及总符合率均为100%。本研究首次建立了用于检测KHV的RAA-LFD方法,该方法特异性强、敏感性高、重复性好、操作方便,可用于KHV的临床样品检测,为KHV现地检测提供了一种简便、快捷的技术手段。In this study,we developed a simple and rapid method for in situ detection of koi herpesvirus(KHV)by combing the advantages of recombinase-aid amplification(RAA)and lateral flow dipstick(LFD)technology,so-called RAA-LFD.According to RAA primer design principle,5 pairs of primers were designed within the highly conserved region of KHV Sph gene,and the No.5 primers were selected as the optimal ones,which were labeled with biotin and FAM at the 5'-end of the reverse-primer and probe,respectively.The reaction time and temperature of RAA were optimized by using the square matrix method,and the results showed that RAA-LFD could effectively amplify the target gene fragment of KHV at a constant temperature of 34℃-40℃for 10 minutes,which allowed an on-site observation.Therefore,a RAA-LFD detection method for KHV was preliminarily established.The specificity test results showed that RAA-LFD just specifically reacted with the nucleic acid of HKV,and had no cross-reaction with that of other poultry pathogens such as cyprini edema virus(CEV),carassius carassius hematopoietic organ necrosis virus(CHNV),epidemic hematopoietic organ necrosis virus(EHNV)and frog rainbow virus(BIV).The RAA-LFD had a comparable sensitivity to the industry conventional PCR,with a minimum detection limit of 50 copies/μL for the plasmid pEASY-KHV samples.The repeatability test results showed that 5 groups of independent pEASY-KHV samples were detected as KHV positive by the RAA-LFD,even though the samples were placed at room temperature for 7 days,indicating that the RAA-LFD has good repeatability.The results from 60 clinical samples detected by both RAA-LFD and conventional PCR showed 100%coincidence between the two methods,including the coincidence rate of negative and positive and the total coincidence.Taken together,the RAA-LFD approach established in this study showed highly specificity,sensitivity and repeatability,which can be applied for rapid clinical diagnosis of KHV.

关 键 词：锦鲤疱疹病毒 重组酶介导扩增 侧向流试纸条 快速检测 

分 类 号：S852.6[农业科学—基础兽医学]