双重PCR快速鉴别无乳链球菌和海豚链球菌  

Rapid identification of Streptococus agalactiae and Streptococus iniae with duplex PCR assay

作　　者：黎炯[1,2] 叶星[1] 卢迈新[1] 邓国成[1] 田园园[1] 江小燕[1] 黎坚平 

机构地区：[1]中国水产科学研究院珠江水产研究所,广东广州510380 [2]上海海洋大学水产与生命学院,上海201306 [3]广东省水产技术推广站,广东广州510380

出　　处：《湖南农业大学学报（自然科学版）》2010年第4期449-452,共4页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家科技支撑计划项目(2006BAD01A1201);现代农业产业技术体系建设专项基金(Nycytx-48-4);广东省农业重点项目(2009B020201003);公益性行业(农业)科研专项(3-49);广东省海洋渔业科技推广专项(A200899B02);农业部"引进国际先进农业科学技术"项目(2010-C26);广东省重大科技兴海(兴渔)项目(070103)

摘　　要：根据无乳链球菌的cfb基因和海豚链球菌16SrRNA基因序列,设计、合成2对引物,优化扩增条件,建立了快速鉴别无乳链球菌和海豚链球菌的双重PCR方法.用该方法扩增无乳链球菌和海豚链球菌,可分别获得474、296bp的特异性片段,扩增嗜水气单胞菌、铜绿假单胞菌等其他常见鱼病原菌无特异性片段.该方法可实现对无乳链球菌和海豚链球菌的快速鉴别,具较高的灵敏度,可检测到基因组含量分别为3.2×10-3ng/μL的无乳链球菌和3.0×10-2ng/μL的海豚链球菌.对采集自广东与海南两省养殖罗非鱼病鱼的11份病原样品进行检测,均可获得474bp片段,测序与BLAST分析结果表明,扩增到的序列均为无乳链球菌cfb基因序列,可从分子水平上确定这些样品为无乳链球菌,与生化鉴定结果一致.Streptococus agalactiae and S. iniae are two common pathogens of fish Streptococcus diseases. Symptoms of disease caused by both Streptococcus pathogens were quite similar, and it was difficult to distinguish the two pathogens from each other directly from the symptoms. Therefore it is necessary to develop a rapid identification method in order to control the disease effectively and rapidly. According to the cfb gene sequence of S. agalactiae and 16S rRNA gene sequence of S. iniae, two pairs of primers were designed and synthesized, and a rapid duplex PCR assay for identification of S. agalactiae and S. iniae was established by optimization of amplification conditions. A 474 bp fragment specific for S. agalactiae and a 296 bp fragment for S. iniae were produced, but no product was amplified from the other common pathogenic bacteria of fish such as Aeromonas hydrophila and Pseudomonas aeruginosa. The method is quite sensitive, and can detect a template concentration as low as 3.2×10-3 ng/μL for S. agalactiae and 3.0×10-2 ng/μL for S. iniae, respectively. A total of eleven pathogen samples collected from pond-cultured tilapia in Guangdong and Hainan provinces were analyzed by duplex PCR. Sequencing and BLAST analysis revealed that all the sequences were cfb gene of S. agalactiae. Results of molecular identification were consistent with the previous results of biochemical assays. The double PCR method would provide a sensitive and accurate method for rapid identification of S. agalactiae and S. iniae.

关 键 词：双重PCR 无乳链球菌 海豚链球菌 cfb基因 16SrRNA 

分 类 号：S941.4[农业科学—水产养殖]