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| 嗜水气单胞菌（Aeromonas hydrophila）的核酸适配体的SELEX筛选及其应用检测 |
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白 月1, 赵玲敏1, 林茂1, 黄力行1, 鄢庆枇1, 王家恩2, 翁齐彪2, 郑 江1
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1.海水养殖生物育种全国重点实验室;2.国家鳗鱼加工技术研发分中心福州
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| 摘要: |
| 嗜水气单胞菌会感染鱼类等水生动物，导致出血性败血症、溃疡病及肠炎等严重疾病，给水产养殖业造成重大经济损失。对该菌进行准确、灵敏的检测，是其病害防治的基础。本文采用SELEX筛选技术获得了嗜水气单胞菌的特异性核酸适配体AH4，该适配体对嗜水气单胞菌的亲和力显著高于迟钝爱德华氏菌、变形假单胞菌、鳗弧菌、哈维氏弧菌等非目标菌（P＜0.05）。在此基础上，建立了针对该菌的适配体-qPCR定量检测方法，该法在10～10? CFU/mL范围内有较好的线性关系，线性拟合系数R2=0.9904，最低检测限达10 CFU/mL，利用该方法对加标海水样品和组织样品中的嗜水气单胞菌进行了检测，证明了该方法的可行性。文中还研究了qPCR法对适配体亲和力的测定，并采用qPCR法测定了AH4对嗜水气单胞菌的亲和常数Kd=171.60±33.15 nmol/L。相关研究成果对嗜水气单胞菌的病害防控及其检测技术的应用开发都具有重要意义。 |
| 关键词: 嗜水气单胞菌（Aeromonas hydrophila） 核酸适配体 SELEX 实时荧光定量qPCR 检测限 |
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| SELECTION OF APTAMER AGAINST AEROMONAS HYDROPHILA BY SELEX AND ITS DETECTION APPLICATION |
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BAI Yue1, ZHAO Ling-Min1, LIN Mao1, HUANG Li-Xing1, YAN Qing-Pi1, WANG Jia-En2, WENG Qi-Biao2, ZHENG Jiang1
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1.State Key Laboratory of Mariculture Breeding;2.National Eel Processing Technology Research and Development Subcenter (Fuzhou)
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| Abstract: |
| Aeromonas hydrophila can infect fish and many other aquatic animals, leading to severe diseases such as hemorrhagic septicemia, ulcer disease, and enteritis, thereby causing substantial economic losses to the aquaculture industry. Accurate and sensitive detection of the pathogen is essential for its disease prevention and control. In the present paper, an aptamer (AH4) capable of specifically recognizing A. hydrophila was selected by SELEX. AH4 exhibited significantly higher affinity to A. hydrophila than to the non-target bacteria including Vibrio anguillarum, Edwardsiella tarda, Pseudomonas plecoglossicida, and Vibrio harveyi (P < 0.05). Based on AH4, an aptamer–qPCR quantitative detection method was developed. The assay showed a good linear relationship in the range of 10–108 CFU/mL, with a linear regression coefficient (R²) of 0.9904 and a limit of detection of 10 CFU/mL. The method was further validated by detecting A. hydrophila in both spiked seawater and tissue samples, confirming its feasibility. The study also investigated the affinity measurement of the aptamer by qPCR, and the affinity constant (Kd) of AH4 for A. hydrophila was determined by qPCR to be 171.60 ± 33.15 nmol/L. These results are of great significance for the detection of A. hydrophila and the prevention and control of its associated diseases. |
| Key words: Aeromonas hydrophila aptamer SELEX real-time quantitative PCR (qPCR) limit of detection |