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›› 2014, Vol. 36 ›› Issue (4): 253-257.

• 论文与报告 •    下一篇

类志贺邻单胞菌间接ELISA检测方法的建立

张新艳   

  1. 福建省淡水水产研究所
  • 收稿日期:2014-06-04 修回日期:2014-06-24 出版日期:2014-08-25 发布日期:2014-08-20
  • 通讯作者: 张新艳
  • 基金资助:
    福建省科技计划项目——省属公益类科研院所基本科研专项;现代农业产业技术体系建设专项

Establish of indirect ELISA detection for Plesiomonas shigelloides

Xin-Yan ZHANG   

  • Received:2014-06-04 Revised:2014-06-24 Online:2014-08-25 Published:2014-08-20
  • Contact: Xin-Yan ZHANG

摘要: 自患病草鱼分离的类志贺邻单胞菌菌株LCCiL90625NA甲醛灭活后注射新西兰大白兔,制备兔抗血清,Proetein A亲和层析柱分离纯化抗体,通过优化反应条件,建立类志贺邻单胞菌间接ELISA检测方法。结果表明,制备的兔抗类志贺邻单胞菌多克隆抗体效价为1:1.28×105,建立的ELISA检测方法可特异性地检测类志贺邻单胞菌,纯化后的多克隆抗体与副溶血弧菌、创伤弧菌、溶藻弧菌、非O-1霍乱弧菌、嗜水气单胞菌、豚鼠气单胞菌、温和气单胞菌、易损气单胞菌和杀鲑气单胞菌均无交叉反应,检测灵敏度为1.0×104 CFU?mL-1。该方法的建立为类志贺邻单胞菌的快速检测和该病的早期诊断及防控提供基础。

关键词: 类志贺邻单胞菌, 多克隆抗体, ELISA检测方法, Plesiomonas shigelloides, polyclonal antibody, ELISA

Abstract: Plesiomonas shigelloides strain LCCiL90625NA, isolated from diseased Ctenopharyngodon idellus, was used for rabbit-serum preparation by injecting the formalin-inactivated bacteria. The rabbit immunoglobulin G was purified by Protein A column. An indirect ELISA was developed for detection of LCCiL90625NA, using rabbit polyclonal antibodies against P. shigelloides as detecting antibody, the titers of that could reach to 1﹕1.28×105. Indirect ELISA assay indicated that the polyclonal antibody had a specific and sensitivity detection for P. shigelloides. The ELISA had no cross-reaction with Vibrio parahaemolyticus, V. vulnificus, V. alginolyticus, V. cholera non O-1, Aeromonas hydrophila, A. caviae, A. sobria, A. trota and A. salmonicida,and the minimum amount of P. shigelloides could be detected by this ELISA, was 1.0×104 CFU?mL-1. The results revealed that the ELISA method was quick,sensitive and repeatable,which was applicable to the detection of P. shigelloides in Ctenopharyngodon idellus.