中文摘要：

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Eu~（3＋）-链霉亲和素作为示踪物,Eu~（3＋）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1.0×10~2 cfu/mL,在1.0×10~1.0×10~6 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.

英文摘要：

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 B 18 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 lgG was incubated with the strain B 18, 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 B 18. This assay showed a good relationship be- tween 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 B 18 with the correlation coeffi- cient 0.9716. The strain B18 was positive in BAS-TRFIA, while 19 other contrast strains ofAeromonas eucrenophila, Aer- omonas jandaei, Aeromonas enteropelogenes, Escherichia coli, Aeromonas bestiarum and Aeromonas media etc. were nega- tive 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 ~C 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-TRFI