杀鲑气单胞菌的实时定量PCR检测方法的建立和应用  

Development and application of real-time PCR assay for detecion of Aeromonas salmonicida

作　　者：刘宗晓[1] 刘荭[2] 史秀杰 高隆英[2] 岳志芹[3] 吕建强[2] 何俊强[2] 江育林[2] 谢从新[1] 

机构地区：[1]华中农业大学水产学院,武汉430070 [2]深圳出入境检验检疫局,518001 [3]山东出入境检验检疫局,青岛266002

出　　处：《海洋水产研究》2008年第5期83-88,共6页Marine Fisheries Research

基　　金：国家质检总局2006年科研项目(2006IK003)资助

摘　　要：选取杀鲑气单胞菌A层A蛋白(VapA)保守序列,利用Primer Express 2.0软件设计引物和探针。以ATCC标准株10倍系列稀释,通过血球计数板计数后进行实时定量PCR,制作标准曲线。通过SDS软件获得细菌数量(X)与Ct值的关系为:Ct=-3.1574lgX+43.6841(相关系数R2=0.992)。实时定量PCR的检测限为6个细菌。建立的方法对杀鲑气单胞菌典型株和非典型株具有较好的特异性,与其他细菌没有交叉反应。杀鲑气单胞菌实时定量PCR方法的建立,对于快速口岸检疫、临床诊断和疫病监测具有重要的应用价值。A method was established for the detection of Aerornonas salrnonicida based on real-time quantitive polymerase chain reaction （qPCR）. A pair of primers and a Taqman probe were designed that are specific for the recognition of a conservative region in the A. salmonicida genome. The real-time PCR had a detection limit of six bacteria. The primer pairs and probe were specific to the A. salmonicida（including typical and atypical strains） and did not cross-react with other bacteria. A standard curve was developed by the SDS software, showing the relationship between the quantity of bacteria（X） and its Ct value, which can be described as Ct=-3. 157 4 lgX＋43. 6841（R^2=0. 992）. This assay had a broad application for basic and clinicalinvestigations.

关 键 词：杀鲑气单胞菌 实时定量PCR TAQMAN探针 

分 类 号：Q93-332[生物学—微生物学] S945[农业科学—水产养殖]