基于核酸适配体的SYBR Green I qPCR法检测鳗弧菌(Vibrio anguillarum)  

A SYBR GREEN I-AND APTAMER-BASED qPCR METHOD FOR VIBRIO ANGUILLARUM DETECTION

作　　者：谭英 赵玲敏[1] 翁齐彪 黄力行[1] 鄢庆枇[1] 黄将远 白月 郑江[1] TAN Ying;ZHAO Ling-Min;WENG Qi-Biao;HUANG Li-Xing;YAN Qing-Pi;HUANG Jiang-Yuan;BAI Yue;ZHENG Jiang(State Key Laboratory of Mariculture Breeding,Engineering Research Center of the Modern Technology for Eel Industry,Ministry of Education,Fisheries College of Jimei University,Xiamen 361021,China;Key Laboratory of Eel Aquaculture and Processing of Fujian Province,Changle Juquan Food Co.Ltd.,Fuzhou 350200,China)

机构地区：[1]海水养殖生物育种全国重点实验室、鳗鲡现代产业技术教育部工程研究中心、集美大学水产学院,福建厦门361021 [2]福建省鳗鱼养殖与加工重点实验室、长乐聚泉食品有限公司,福建福州350200

出　　处：《海洋与湖沼》2024年第4期942-950,共9页Oceanologia Et Limnologia Sinica

基　　金：福建省自然科学基金项目,2021J01823号,2023J01762号;厦门市科技补助项目,2023CXY0302号;鳗鲡现代产业技术教育部工程研究中心开放基金项目,RE202308号。

摘　　要：鳗弧菌(Vibrio anguillarum)可感染鲈鱼、鳗鲡等多种水产养殖动物,是水产养殖中的重要病原菌,对其进行快速检测是病害防控的基础。利用鳗弧菌与其核酸适配体间较强的亲和特异性,通过核酸适配体来识别、结合鳗弧菌,然后以结合的核酸适配体为模板,进行SYBR Green I实时荧光定量PCR(qPCR)扩增,通过Ct值来定量检测鳗弧菌的浓度,从而建立了鳗弧菌的适配体-qPCR定量检测方法。从特异性、标准曲线、灵敏度、重复性和应用效果对该方法进行分析,表明该方法具有很强的特异性,能特异性地扩增鳗弧菌,且对哈维氏弧菌、溶藻弧菌、变形假单胞菌、大肠杆菌、嗜水气单胞菌和迟钝爱德华氏菌均无扩增;在10^(3)~10^(11) CFU/L的检测范围内有较好的线性关系,可用于鳗弧菌的定量检测;同时,该方法有较高的灵敏度和稳定性,其最低检测限为10^(3) CFU/L,组内和组间变异系数分别小于0.17%和1.98%;最后采用该方法对鱼体组织样品进行了应用检测,证明了该方法具有较好的可行性和应用性,可用于水产品或食品中鳗弧菌的定量检测。Vibrio anguillarum is an important bacterium pathogen in aquaculture,and can infect many aquaculture animals such as perch and eel.A rapid detection of the pathogen is the basis for the prevention and control of the disease caused by the bacterium.Considering the strong affinity and good specificity between V.anguillarum and its aptamer,the aptamer was used to identify and bind to the bacterium,and then acted as a template for SYBR Green I real-time fluorescence quantitative PCR(qPCR)amplification.The concentration of V.anguillarum can be quantitatively detected by the Ct value obtained from the qPCR.Consequently,an aptamer-qPCR quantitative assay method for V.anguillarum was established.The specificity,standard curve,sensitivity,repeatability,and application of the method were studied.It was found that the method had good specificity and could specifically amplify V.anguillarum,but did not amplify V.harveyi,V.alginolyticus,Pseudomonas plecoglossicida,Escherichia coli,Aeromonas hydrophila,and Edwardsiella tarda.The detection method has good linear relationship in the range of 10^(3)-10^(11) CFU/L,which can be applied for the quantitative detection to V.anguillarum.Also,the aptamer-qPCR method had high sensitivity and stability,in which the lowest detection limit was 10^(3) CFU/L and the coefficients of variation(CV)of intra-and inter-group were less than 0.17%and 1.98%,respectively.Finally,the method was applied to detect V.anguillarum in fish tissues,and proved to be feasible and applicable.The aptamer-qPCR method established in this study can be used for quantitative detection of V.anguillarum in aquatic products and food.

关 键 词：鳗弧菌 核酸适配体 实时荧光定量PCR 检测限 

分 类 号：S947[农业科学—水产养殖] Q939[农业科学—水产科学]