摘要
根据水产养殖中常见的3种病原菌鳗弧菌(Vibrio anguillarum)、嗜水气单胞菌(Aeromonas hydrophila)及迟缓爱德华氏菌(Edwardsiella tarda)的研究资料,筛选3个毒力相关基因toxR、aerA、evpA设计引物和探针,构建简型基因芯片,并使用对虾白斑病综合征病毒(WSSV)variable region的PCR荧光标记产物作为表面化学质控。通过已构建和优化的多重PCR反应条件,获得了3个目的基因的PCR产物;经过芯片制作过程的优化,将探针以终浓度20μmol/L溶于50%DMSO,在室温、相对湿度45%的条件下点印于醛基基片表面。扩增的PCR产物与杂交液混合后,在42℃杂交2 h就可检测到理想的杂交信号。芯片的灵敏度试验结果表明,可以检测到的3种水产病原菌的最低模板DNA为:鳗弧菌3×102拷贝、嗜水气单胞菌5×103拷贝、迟缓爱德华氏菌6×101拷贝。该芯片可成功应用于患病大菱鲆内脏的细菌分离物的鉴定。
Based on the reported information of three major aquatic pathogens Vibrio anguillarum,Aeromonas hydrophila,and Edwardsiella tarda,three primer pairs and oligo probes were designed against the gene toxR,aerA and evpA to construct a simple and convenient multiplex PCR-microarray.The gene variable region of shrimp white spot syndrome virus（WSSV） was used as the surface chemistry quality control on the microarray.The target products which were amplified by multiplex PCR were hybridized to the microarray.The optimized preparation protocol employed an Aldehyde slide and 20 μmol/L amino-group DNA probes in 50% DMSO to be spotted on the slide at 45% humidity.The prepared microarray slide was hybridized with fluorescent PCR products at 42 ℃ for 2 h.The overall LDLs（lowest detection level） of the microarray were tested with the genomic DNA of three bacterial strains around 3×102 copies of V.anguillarum,5×103 copies of A.hydrophila,and 6×101 copies of E.tarda.The technology was successfully demonstrated in the identification of bacterial isolates from a diseased turbot.
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第3期37-42,共6页
Periodical of Ocean University of China
基金
农业公益性行业科研专项(200803012)
国家现代产业技术体系建设(nycytx-46)
国家高技术研究发展计划项目(2006AA100306)
基本科研业务费专项资金项目(2007-GY-03)资助
关键词
基因芯片
水产病原菌
检测
microarray
aquatic bacterial pathogens
establishment
