时间分辨荧光免疫分析高灵敏检测嗜水气单胞菌

doi:10.6023/A16110619

化学学报 ›› 2017, Vol. 75 ›› Issue (4): 398-402.DOI: 10.6023/A16110619 上一篇下一篇

研究论文

时间分辨荧光免疫分析高灵敏检测嗜水气单胞菌

林鹏, 卢洁, 郭松林, 冯建军, 王艺磊, 陈锦民

集美大学水产学院农业部东海海水健康养殖重点实验室厦门 361021

投稿日期:2016-11-21 发布日期:2017-02-23
通讯作者: 林鹏,E-mail:linpeng@jmu.edu.cn;Tel.:0592-6181420;Fax:0592-6181476 E-mail:linpeng@jmu.edu.cn
基金资助:
项目受国家自然科学基金（No.31272685）资助.

Sensitive Detection of Aeromonas Hydrophila by Time-Resolved Fluorimmunoassay

Lin Peng, Lu Jie, Guo Songlin, Feng Jianjun, Wang Yilei, Chen Jinmin

Fisheries College, Jimei University, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Xiamen 361021

Received:2016-11-21 Published:2017-02-23
Contact: 10.6023/A16110619 E-mail:linpeng@jmu.edu.cn
Supported by:
Project supported by the National Natural Science Foundation of China (No. 31272685).

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摘要/Abstract

建立了生物素-链霉亲和素时间分辨荧光免疫分析（BAS-TRFIA）高灵敏检测嗜水气单胞菌的新方法.以兔抗IgG包被微孔板，加入嗜水气单胞菌菌株B18和生物素化IgG，生成的夹心复合物用Eu³⁺-链霉亲和素作为示踪物，Eu³⁺可与离解增强液形成时间分辨荧光（TRF），通过TRF值对病原菌定量.方法的检测限为1.0×10² cfu/mL，在1.0×10~1.0×10⁶ cfu/mL范围内线性良好，相关系数0.9716.用该法检测时，不同来源的嗜水气单胞菌以及其它气单胞菌均呈阴性.板内和板间的变异系数分别小于5.00%和9.00%.所有相关检测试剂37℃恒温放置6 d后，检测性能无明显改变.对60份人工感染的美洲鳗鲡组织样品，包括肝、肾、肠、鳃和肌肉等组织进行检测，阳性检测率为98.33%.结果表明，BAS-TRFIA法检测嗜水气单胞菌，灵敏度高、特异性好、操作简便，该方法为水产养殖病原菌检测提供了新的思路.

关键词: 时间分辨荧光免疫分析, 嗜水气单胞菌, 生物素-亲和素系统, 美洲鳗鲡, 水产养殖

The aim of this study is to establish a biotin-avidin system and time-resolved fluorimmunoassay (BAS-TRFIA) for quantitative analysis of Aeromonas Hydrophila. First, A. Hydrophila strain B18 isolated from diseased Anguilla rostrata, was used for rabbit anti-serum preparation by injecting the formalin-inactivated bacteria. The rabbit immunoglobulin (IgG) was purified by SPA affinity column. Then, a 96-well microtiter plate precoated with the rabbit IgG was incubated with the strain B18, and biotinylated IgG was pipette to the wells to form a typical double-antibody-sandwich immune complex. Finally, Eu³⁺-labeled streptavidin was added the wells to produce affinity reaction. Time-resolved fluorescence (TRF) of Eu³⁺ was measured in enhancement solution to reflect the quantity of the strain B18. This assay showed a good relationship between TRF and the concentration of the strain B18. The limit of detection (LOD) was 1.0×10² cfu/mL, and the LOD of BAS-TRFIA was improved 400-fold compared to the already reported ELISA. The established sandwich BAS-TRFIA showed a good sixth order polynomial fitting from 1.0×10 to 1.0×10⁶ cfu/mL for the strain B18 with the correlation coefficient 0.9716. The strain B18 was positive in BAS-TRFIA, while 19 other contrast strains of Aeromonas eucrenophila, Aeromonas jandaei, Aeromonas enteropelogenes, Escherichia coli, Aeromonas bestiarum and Aeromonas media etc. were negative in the assay, indicating that the antibody had high specificity. The intra-assay and inter-assay standard deviation were less than 5.00% and 9.00%, respectively. The TRF didn't change obviously after the related reagents were placed at 37℃ for 6 days. The method was used to detect the tissues including liver, kidney, intestine, gill and muscle from A. rostrata infected artificial with the strain B18. The result showed 98.33% of 60 samples were positive. BAS-TRFIA for A. hydrophila strain B18 was sensitive, specific and simple. The results indicate that the established BAS-TRFIA has potential application for screening A. hydrophila in aquaculture.

Key words: time-resolved fluorimmunoassay, Aeromonas hydrophila, biotin-avidin system, Anguilla rostrata, aquaculture

引用此文

林鹏, 卢洁, 郭松林, 冯建军, 王艺磊, 陈锦民. 时间分辨荧光免疫分析高灵敏检测嗜水气单胞菌[J]. 化学学报, 2017, 75(4): 398-402.

Lin Peng, Lu Jie, Guo Songlin, Feng Jianjun, Wang Yilei, Chen Jinmin. Sensitive Detection of Aeromonas Hydrophila by Time-Resolved Fluorimmunoassay[J]. Acta Chim. Sinica, 2017, 75(4): 398-402.

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时间分辨荧光免疫分析高灵敏检测嗜水气单胞菌

Sensitive Detection of Aeromonas Hydrophila by Time-Resolved Fluorimmunoassay

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