舒伯特气单胞菌TaqMan-MGB实时荧光定量PCR检测方法的建立及应用  

TaqMan-MGB Probe real-time Fluorescence Quantitative PCR method for detection ofAeromonas schubertii

作　　者：刘春[1] 曹际振 张德锋[1] 常藕琴[1] 李凯彬[1] 王芳[1] 姜兰[1] 王庆[1] LIU Chun;CAO Jizhen;ZHANG Defeng;CHANG Ouqin;LI Kaibin;WANG Fang;JIANG Lan;WANG Qing(Key Laboratory of Fishery Drug Development of Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province,Pearl River Fiskeries Research Institute,Chinese Academy of Fishery Scienees,Guangzhou 510380,China)

机构地区：[1]中国水产科学研究院珠江水产研究所,农业部渔药创制重点实验室,广东省水产动物免疫技术重点实验室,广东广州510380

出　　处：《珠江水产科学》2018年第4期1-8,共8页Finsheries Science and Technology Industry

基　　金：广东省科技计划项(2016A030303028,2017A040403008);现代农业产业技术体系建设专项资金(CARS-46);中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金(2017HY-ZC0406).

摘　　要：为了在发病早期灵敏、特异地快速检测水产养殖过程中的舒伯特气单胞菌(Aeromonas schubertii),本研究建立了rpoD基因的TaqMan-MGB实时荧光定量PCR检测方法,并对该方法的灵敏度、可重复性和特异性进行评价。结果显示:建立的荧光定量PCR检测方法标准曲线有较好的线性关系;每微升最低可以检测到18个rpoD基因拷贝;30个平行样品重复性试验组内变异系数为0.44%;对其它11种水产养殖常见细菌均无扩增反应。应用该方法对池塘水和鱼体组织中的舒伯特气单胞菌含量检测结果与活菌计数结果有较好的一致性。研究表明,建立的TaqMan-MGB实时荧光定量PCR方法灵敏度高、特异性强,能较好的用于养殖水环境和鱼体组织中舒伯特气单胞菌的检测,对舒伯特气单胞菌病的早期诊断、流行病学研究和防控技术研究等具有重要意义。The purpose of this study is to establish a high sensitivity and specificity for the rapid method to detect A. schubertii in aquiculture.According to the rpoD gene sequence of A.schubertii available in GenBank database,a pair of specific primer and TaqMan-MGB probes was designed for establishing a TaqMan-MGB real-time fluorescence quantitative PCR method to detect A.schubertii.The sensitivity,repeatability and specificity of the method were evaluated.The results showed that the TaqMan-MGB real-time fluorescence quantitative PCR method was established and the standard curve showed a good linear relationship.Sensitivity tests showed that the detection limit was 18 copies ·μL^-1.Results of repeatability tests showed that the coefficient of variation was 0.44%,indicating that this method had high repeatability.Furthermore,the method had high specificity for A.schubertii without cross reactions with templates from other aquatic bacteria.In clinical samples tests,the detection results of A.schubertii in pond water and fish tissues by established fluorescence PCR were consistent with the results of live bacteria counting.The TaqMan-MGB real-time fluorescence quantitative PCR method was demonstrated to be specific, sensitive and simple for the rapid detection and quantification of A.schubertii.

关 键 词：鳢科鱼类 舒伯特气单胞菌 rpoD基因 TaqMan-MGBreal-timePCR 检测方法 

分 类 号：S944.12[农业科学—水产养殖]