嗜水气单胞菌BA-ELISA的建立及其在病原检测中的应用  

Establishment and Application of Biotin-avidin-amplified Enzyme-linked Immunosorbent Assay on Aeromonas hydrophila of Chinese Soft-shelled Turtles,Pelodiscus sinensis

作　　者：胡大雁[1] 钱冬[2] 刘问[2] 张友平[2] 潘清清[1] 陈昌福[1] 

机构地区：[1]华中农业大学水产学院,武汉430070 [2]浙江省淡水水产研究所,湖州313001

出　　处：《华中农业大学学报》2008年第1期80-85,共6页Journal of Huazhong Agricultural University

基　　金：“十一五”国家攻关课题(2006BAD03B0502)资助

摘　　要：从中华鳖中分离的嗜水气单胞菌致病株T0608经福尔马林灭活后,注射免疫新西兰兔,制备了高价免疫血清,血清的菌体凝集效价可达1/1 280;G蛋白亲和层析纯化兔IgG,将IgG用生物素标记,建立了生物素-亲和素双抗体夹心酶联免疫吸附法(BA-ELISA);其最低检出限为2.5×103cfu,相当于0.5 ng菌体蛋白,其检测灵敏度为ABC-ELISA(4.0×104cfu)和间接ELISA(4.0×104cfu)的16倍;特异性试验结果表明,本法与鱼类气单胞菌、拟态弧菌、海豚链球菌、大肠杆菌等无交叉反应。BA-ELISA可不经细菌培养直接检测人工感染48 h中华鳖的肝、肠道组织中的嗜水气单胞菌。用该方法对湖州市龟鳖养殖场的水样、中华鳖和巴西龟肝、肠道内容物进行检测,结果表明该方法可较好地应用于发病中华鳖的病原早期诊断及养殖中华鳖的病原检测。Aeromonas hydrophila T0608,isolated from diseased Chinese soft-shelled turtles,Pelodiscus sinensis,was used for rabbit anti-serum preparation by injecting the formalin-inactivated bacteria.The agglutination titer of rabbit serum was 1/1 280.The rabbit immunoglobulin G was purified by protein G column and labeled with biotin.A biotin-avidin-amplified enzyme-linked immunosorbent assay（BA-ELISA） was developed for detection of T0608.The sensitivity of ABC-ELISA,indirect ELISA and BA-ELISA was compared.The detection limit of BA-ELISA was Approximately 2.5×10^3 cfu,or 0.5 ng bacterial protein,16 times more sensitive than ABC-ELISA and indirect ELISA.No cross-reaction was observed when fish A.hydrophila BSK-10,Vibrio mimicus,Streptococcus iniae and E.coli were applied instead of T0608.BA-ELISA can be directly used to detect aeromonads in liver and intestine of Chinese soft-shelled turtles 48 h after challenged with T0608.This method can be used to detect aeromonads in water,intestine and liver samples of Chinese soft-shelled turtles and red-eared slider turtles,which was cultivated in alkaline peptone broth for 12 hours.BA-ELISA can be considered as a useful method for early diagnosis of Chinese soft-shelled turtles infected with aeromonads,as well as aeromonads detection in Chinese soft-shelled turtles farms.

关 键 词：生物素-亲和素双抗体夹心酶联免疫吸附法 中华鳖 嗜水气单胞菌 检测 

分 类 号：S947.12[农业科学—水产养殖]