Acta Chim. Sinica ›› 2017, Vol. 75 ›› Issue (4): 398-402.DOI: 10.6023/A16110619 Previous Articles     Next Articles

Article

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   

  1. 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
  • 收稿日期:2016-11-21 出版日期:2017-04-15 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   

  1. Fisheries College, Jimei University, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Xiamen 361021
  • Received:2016-11-21 Online:2017-04-15 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).

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