鳗鲡源创伤弧菌的间接ELISA快速检测

The Indirect ELISA for Rapid Detection of Vibrio vulnificus from Eels

创伤弧菌(Vibrio vulnificus)血清2型对鳗鲡有专一的感染性,是鳗鲡出血性败血症、烂鳃病和烂尾病的重要病原菌。采用间接ELISA法对制备的兔抗创伤弧菌多克隆抗血清进行效价测定,结果表明其效价高达1∶512000。从发病鳗鲡中分离出创伤弧菌和37株其他致病菌,将38株病原菌以较低浓度(1×106cfu/mL)包被后通过兔抗创伤弧菌血清进行交叉反应水平检测,结果表明,兔抗创伤弧菌血清稀释到1∶256000或1∶128000时,其与37株其他病原菌均未产生明显的ELISA交叉反应。该血清分别经1∶64000和1∶32000稀释后,采用ELISA方法检测了经创伤弧菌、鳗鲡爱德华氏菌(Edwardsiella anguillarum)和嗜水气单胞菌(Aeromonas hydrophila)攻毒感染死亡的美洲鳗鲡(Auguilla rostrata)脏器组织悬液(肝脏、肾脏和鳃),结果表明,兔抗创伤弧菌血清经1∶32000稀释后可特异性检出鳗鲡鳃和肾脏组织中的创伤弧菌。

Serum type 2 of Vibrio vulnificus is highly pathogenic to eel and cause hemorrhagic sepsis,gill rot and tail rot.In this study,V.vulnificus and 37 strains of pathogenic bacteria isolated from infected eel were used as bacterial strains.The polyclonal antiserum titers of rabbit anti-V.vulnificus were determined by indirect ELISA,and the results showed that the titer of the antiserum was 1∶512000.Thirty-eight strains of pathogenic bacteria were coated with the concentration of 1×106 cfu/mL and then cross-reacted with the antiserum.The results showed that when the antiserum was diluted to 1∶256000 or 1∶128000,there was no obvious ELISA cross-reaction between the V.vulnificus and other 37 bacterial pathogens in eels.After the antiserum was diluted at 1∶64000 and 1∶32000 respectively,the suspensions of liver,kidney and gill of Auguilla rostrata infected by V.vulnificus,E.anguillarum and A.hydrophila were detected by ELISA.The results showed that the 1∶32000 diluted antiserum specifically detect the bacterial pathogen of V.vulnificus in the gill and kidney of eels.