基于电化学生物传感技术的鱼类IHNV病毒检测方法构建  

Development of an Efficient Method for Detecting IHNV Based on Electrochemical Biosensing Technique

作　　者：赵紫霞[1] 许建[1] 张研[1] 江炎亮 陈强[2] 

机构地区：[1]中国水产科学研究院农业部水生动物基因组学重点实验室/渔业生物技术北京市重点实验室,北京100141 [2]南开大学生命科学学院/生物活性材料教育部重点实验室,天津300071

出　　处：《农业生物技术学报》2018年第2期357-364,共8页Journal of Agricultural Biotechnology

基　　金：国家科技支撑计划项目(No.2015BAD25B01)

摘　　要：本研究旨在开发传染性造血器官坏死病毒(Infectious hematopoietic necrosis virus,IHNV)现场检测新技术,用于鉴定鱼类苗种是否携带IHNV,降低疫病传播风险,实现水产健康养殖管理。基于IHNV基因组序列保守性分析结果,以保守性最高的基质蛋白编码基因(matrix,M)为检测靶序列,设计了6条IHNV特异性扩增引物,对样品RNA进行逆转录等温链置换扩增。在部分引物5’端标记二茂铁分子,使扩增产物带有电化学信号,在部分底物中标记生物素分子,使扩增产物被亲和素修饰的电极表面捕获锚定,基于该原理构建了电化学生物传感体系,通过检测扩增产物的循环伏安信号,判断样品中是否携带IHNV。本方法能够在15 min内完成核酸扩增,且扩增反应在59~65℃的宽松温度区间内均可进行,无需精准控温,对设备和操作要求较低。在扩增温度63℃、信噪比为3的条件下,本方法最低检测限(limit of detection,LoD)为6.2 copies/μL,能够对未表现出感染症状的鲑鳟类鱼苗、鱼卵进行IHNV检测,适用于苗种场、养殖场等生产经营性场所的现场检测,在水产苗种鉴定领域具有良好的应用前景。Infectious hematopoietic necrosis virus（IHNV） is an economically important pathogen causing significant mortalities in a wide variety of salmonid species, including the main aquaculture species, Atlantic salmon（Salmo salar） and rainbow trout（Oncorhynchus mykiss）. Aimed to disease control and aquatic biosecurity, In this study, a new electrochemical biosensing method for IHNV on-site detection was developed to identify the IHNV-carrying salmonid fries or eggs. Based on evolutionary conservation analysis of 357 nonredundant IHNV sequences from different origins, the matrix gene（M） was found most conserved and selected as target sequence. Six IHNV specific primers were designed for reverse transcription isothermal amplification, according to M gene sequence of the IHNV Chinese strain Ch20101008. Biotinylated dUTP was mixed into d NTPs in the amplification system, thus amplification products were able to be captured andanchored on avidin modified electrode surfaces. And the 5＇ ends of 2 primers were labeled with ferrocene,which made the amplification products electrochemically active and capable to be detected by cyclic voltammetry（CV）. The amplification for nucleic acid was able to be finished in 15 min, and the current value of oxidation peak had obvious positive correlation with the copy numbers of IHNV in the template. With amplification temperature at 63 ℃ and signal to noise ratio at 3, the limit of detection（Lo D） was calculated to be 6.2 copies/μL, which was capable to distinguish asymptomatic IHNV carriers. Detectable products could be produced across a wide amplification temperature range from 59 to 65 ℃, thus the IHNV detection could be performed without precise thermal control. Therefore, the electrochemical biosensing method has promising application prospects in IHNV detection in aquaculture industry, especially salmonid farms and hatcheries.anchored on avidin modified electrode surfaces. And the 5＇ ends of 2 primers were labeled with ferrocene, which made the amplificat

关 键 词：传染性造血器官坏死病毒(IHNV) 鲑科鱼类 电化学信号 生物传感 

分 类 号：S941.414[农业科学—水产养殖] O657.1[农业科学—水产科学]