十足目虹彩病毒1 RAA-LFD快检方法建立与应用  

Establishment and Application of RAA-LFD Assay for Decapod Iridescent Virus 1

作　　者：田飞焱 吴众怡 徐节华 黄海莉 裴建明 孟霞 刘文珍 周文华 谢世红 Tian Feiyan;Wu Zhongyi;Xu Jiehua;Huang Haili;Pei Jianming;Meng Xia;Liu Wenzhen;Zhou Wenhua;Xie Shihong(Jiangxi Agricultural and Technical Extension Center,Nanchang 330046,Jiangxi,China)

机构地区：[1]江西省农业技术推广中心,江西南昌330046

出　　处：《中国动物检疫》2025年第2期100-107,共8页China Animal Health Inspection

基　　金：江西省大宗淡水鱼产业技术体系病害防控岗项目(2025—2027年);江西省农牧渔业科研指导性课题项目(2021-29,2023-32)。

摘　　要：为建立一种适用于现场检测十足目虹彩病毒1(Decapod iridescent virus 1,DIV1)的方法,利用DIV1 ATPase编码基因保守序列设计RAA-nfo特异性引物和探针,并结合侧向流试纸条(LFD)技术,通过优化反应时间和温度等条件,建立了可现场检测DIV1的RAA-LFD方法。该方法能够在37℃下恒温反应20 min,并在LFD显色5 min后判读结果;对嗜水气单胞菌、弗氏柠檬酸杆菌、白斑综合征病毒(WSSV)、传染性皮下和造血组织坏死病毒(IHHNV)、虾肝肠胞虫(EHP)以及致急性肝胰腺坏死病的副溶血弧菌(Vp_(AHPND))等虾类病原无交叉反应,仅对DIV1检测呈阳性;最低检测限为5.25×10^(1) copies/反应;组内和组间重复性试验结果良好。使用该方法与已报道的套式PCR和qPCR方法对采集的60份克氏原螯虾样品进行平行检测,检测结果与套式PCR一致,与qPCR方法相比,其敏感性和特异性分别为83.3%和100%。结果表明,该方法反应快速、操作简便、灵敏度高、特异性好,具有良好的重复性和可信度,适用于临床样品的现场检测。In order to develop an on-site assay for Decapod iridescent virus 1(DIV1),specific primers and probes for RAA-nfo were designed based on the conserved sequence of the gene encoding DIV1 ATPase to develop RAALFD assay for on-site detection of DIV1 in combination with lateral flow dipstick(LFD)and through optimizing the conditions of reaction time and temperature.The reaction could be completed at 37℃for 20 min,and the results could be interpreted after 5 minutes of LFD coloration;no cross-reaction with other shrimp pathogens was observed,such as Aeromonas hydrophophilus,Citrobcter freundii,white spot syndrome virus(WSSV),infectious hypodermal and haematopoietic necrosis virus(IHHNV),entamoeba hepatica of shrimp(EHP)and Vibrio parahaemolyticus(Vp_(AHPND)),while only DIV1 was tested positive;the minimum detection limit was 5.25×10^(1 )copies/reaction;and the results of intra-and inter-group reproducibility tests were good.60 Procambarus clarkii samples were detected using the assay in parallel with the reported nested PCR and qPCR,the results of the assay were consistent with those using the nested PCR,and the sensitivity and specificity were 83.3%and 100%,respectively,compared with the qPCR.In conclusion,the established assay was rapid,accessible,highly sensitive and specific,with good reproducibility and reliability,and appropriate for on-site detection of clinical samples.

关 键 词：十足目虹彩病毒1 重组酶介导扩增技术 侧流层析技术 恒温扩增 快速检测 

分 类 号：S852.6[农业科学—基础兽医学]