| 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,但对哈维氏弧菌、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103～1011 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103 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。 |
| 关键词: 鳗弧菌 核酸适配体 实时荧光定量PCR 检测限 |
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| 基金项目:福建省自然科学基金项目（2021J01823,2023J01762）；厦门市科技补助项目（NO.2023CXY0302）；鳗鲡现代产业技术教育部工程研究中心开放基金项目（RE202308）。 |
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| Detection of Vibrio anguillarum by SYBR Green I qPCR method based on aptamer |
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TAN Ying1, ZHAO Lin-Min1, WENG Qi-Biao2, HUANG Li-Xing1, YAN Qing-Pi1, HUANG Jiang-Yuan1, BAI Yue1, ZHENG Jiang1
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1.Fisheries College of Jimei University;2.Changle Juquan Food Co,Ltd
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| Abstract: |
| Vibrio anguillarum can infect many aquaculture animals such as perch and eel, and is an important pathogen in aquaculture. Rapid detection of the pathogen is the basis for prevention and control of the disease caused by the bacterium. Based on the strong affinity and good specificity between V. anguillarum and its aptamer, aptamer was utilized to identify and bind to the bacterium, and then used as a template for SYBR Green I real-time fluorescence quantitative PCR 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 Vibrio harveyi, Vibrio alginolyticus, Pseudomonas plecoglossicida, Escherichia coli, Aeromonas hydrophila and Edwardsiella tarda. The detection method has good linear relationship in the range of 103 ~ 1011 CFU/L, which can be used for the quantitative detection of V. anguillarum. The aptamer-qPCR method also has high sensitivity and stability, in which the lowest detection limit is 103 CFU/L and the coefficients of variation (CV) of intra- and inter-group are less than 0.17% and 1.98%, respectively. Finally, the method was applied to detect V. anguillarum in the samples of fish tissues, and proved to be feasible and applicable. The aptamer-qPCR method established in the present paper can be used for quantitative detection of V. anguillarum in aquatic products and food. |
| Key words: Vibrio anguillarum Aptamer Real-time quantitative PCR Limit of deteciton |