黄颡鱼杆套病毒TaqMan qPCR检测方法的建立与应用  

Development and application of TaqMan qPCR detection method for Yellow catfish nidovirus

作　　者：王亿文 潘晓艺[1] 蔺凌云[1] 刘思玉 姚嘉赟[1] 王甘翔 沈锦玉[1,2] WANG Yiwen;PAN Xiaoyi;LIN Lingyun;LIU Siyu;YAO Jiayun;WANG Ganxiang;SHEN Jinyu(Ministry of Agriculture and Rural Areas Key Laboratory of Healthy Freshwater Aquaculture,Key Laboratory of Fish Health and Nutrition of Zhejiang Province,Zhejiang Institute of Freshwater Fisheries,Huzhou 313001,Zhejiang,China;Shanghai Ocean University,Shanghai 201306,China;Pinghu Fisheries Technology Promotion Center,Pinghu 314200,Zhejiang,China)

机构地区：[1]浙江省淡水水产研究所农业农村部淡水渔业健康养殖重点实验室浙江省鱼类健康与营养重点实验室,浙江湖州313001 [2]上海海洋大学,上海201306 [3]浙江省平湖市渔业技术推广中心,浙江平湖314200

出　　处：《淡水渔业》2024年第6期48-56,共9页Freshwater Fisheries

基　　金：湖州市乡村振兴项目(2020ZD2033);浙江省基础公益研究探索项目(ZCLTGN24C1901);浙江省“三农九方”科技协作计划项目(2024SNJF053)。

摘　　要：黄颡鱼杆套病毒(Yellow catfish nidovirus,YCNdV)是近年来在养殖黄颡鱼(Pelteobagrus fulvidraco)中新发现的病毒性病原,为找到灵敏检测该病毒的方法,本研究根据YCNdV的衣壳基因序列设计特异性引物及探针,对反应体系和反应条件进行优化,建立了TaqMan qPCR方法,并与套氏PCR进行了临床样品的检出率比较。结果显示:反应体系优化后的引物和探针终浓度分别为0.4μmol/L和0.1μmol/L,获得的最佳退火延伸温度为58℃,该条件下,最低检测限为4 copies的pMD19-YCNdV.cap质粒标准品;标准曲线线性关系好且线性范围广(3.37×10^(10)~3.37×100 copies/μL),相关系数(R^(2))为0.9984,扩增效率为91.3%;特异性检测结果表明,与黄颡鱼小RNA病毒(Yellow catfish picornavirus,YCPrV)、草鱼呼肠孤病毒Ⅱ型(Grass carp reovirus genotypeⅡ,GCRV-Ⅱ)、鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,CyHV-2)、大口黑鲈弹状病毒(Micropterus salmoides rhabdovirus,MSRV)、大口黑鲈蛙虹彩病毒(Largemouth bass virus,LMBV)、传染性脾肾坏死病毒(Infectious spleen and kidney necrosis virus,ISKNV)、鲤浮肿病毒(Carp edema virus,CEV)和锦鲤疱疹病毒(Koi herpesvirus,KHV)均无交叉扩增信号;重复性实验结果显示,组间和组内变异系数均小于0.6%;对保存的32份疑似黄颡鱼杆套病毒感染样品进行检测,结果表明,TaqMan qPCR检出率较套氏PCR高9.37个百分点;TaqMan qPCR检测发病黄颡鱼各组织发现,在脾、鳃和肾中YCNdV病毒载量显著高于其它组织。以上结果表明,本研究建立的TaqMan qPCR检测方法具有良好的特异性、重复性和超高的灵敏度,可用于YCNdV的定量检测,为YCNdV引起疾病的早防早治提供可靠检测手段。In recent years,yellow catfish nidovirus(YCNdV)is a newly identified viral pathogen in cultured yellow catfish.To provide an accurate and sensitive detection method for YCNdV,this study designed specific primers and probes based on the capsid gene sequence of YCNdV.The reaction system and conditions were optimized,a qPCR method was established along with a standard curve,and the qPCR method’s sensitivity,specificity,and stability were tested.The method was then applied to clinical samples,and its detection rate was compared with that of nested PCR.The results showed that the final optimized concentrations of the primers and probes were 0.4μmol/L and 0.1μmol/L,respectively,with the optimal annealing extension temperature being 58℃.Under these conditions,the minimum detection limit was 4 copies of the pMD19-YCNdV.cap plasmid standard.The generated standard curve demonstrated good linearity and a wide linear range(3.37×10^(10) copies/μL to 3.37×100 copies/μL),with a correlation coefficient(R^(2))of 0.9984 and an amplification efficiency of 91.3%.Specificity testing revealed no cross-amplification signals with yellow catfish picornavirus(YCPrV),grass carp reovirus genotypeⅡ(GCRV-Ⅱ),Cyprinid herpesvirus 2(CyHV-2),Micropterus salmoides rhabdovirus(MSRV),largemouth bass virus(LMBV),infectious spleen and kidney necrosis virus(ISKNV),carp edema virus(CEV),and Koi herpesvirus(KHV).Reproducibility experiments showed that the coefficients of variation were less than 0.6%both between and within groups.Testing 32 stored suspected YCNdV infection samples revealed that the detection rate of TaqMan qPCR was 9.375%higher than that of nested PCR.TaqMan qPCR detection of various tissues in diseased yellow catfish showed significantly higher viral loads of YCNdV in the spleen,gills,and kidneys compared to other tissues.These results indicate that the TaqMan qPCR detection method established in this study possesses excellent specificity,reproducibility,and ultra-high sensitivity,making it suitable for the quantitative de

关 键 词：黄颡鱼(Pelteobagrus fulvidraco) 杆套病毒 TaqMan qPCR 组织分布 

分 类 号：S941.41[农业科学—水产养殖]