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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.
A new method for sensitive detection of Aeromonas hydrophila by biotin-streptavidin time-resolved fluorescence immunoassay (BAS-TRFIA) was established.Using rabbit anti-IgG coated microplate, adding Aeromonas hydrophila Strain B18 and biotinylated IgG, Eu ~ (3 +) - streptavidin were used as tracer for the sandwich complex. Eu ~ (3+) could form time-resolved fluorescence (TRF) with dissociation- The pathogen was quantified by the TRF value.The detection limit of the method was 1.0 × 10 ~ 2 cfu / mL and the linearity was good in the range of 1.0 × 10 ~ 1.0 × 10 ~ 6 cfu / mL with a correlation coefficient of 0.9716. The source of Aeromonas hydrophila and other Aeromonas were negative.The coefficient of variation of in-plate and in-plate were less than 5.00% and 9.00%, respectively.All the detection reagents were not obvious at 6 ℃ for 6 days The positive detection rate was 98.33% in 60 samples of artificially infected Anguilla japonica, including liver, kidney, intestine, gill and muscle.The results showed that BAS-TRFIA method could detect Aeromonas hydrophila, The method has high sensitivity, good specificity and easy operation. The method provides a new idea for the detection of aquaculture pathogens.