罗非鱼海豚链球菌PCR检测方法的建立

Development of a PCR assay for Streptococcus iniae in tilapia

为建立准确快速的海豚链球菌鉴定方法，设计合成了海豚链球菌种特异性引物CM1／CM2，进行了其特异基因片段的PCR扩增、反应条件的优化及方法的特异性和敏感性试验；同时还进行了不同检测材料的比较及9份临床样品检测。结果表明，引物CM1／CM2只能从海豚链球菌中扩增到特异性基因片段，供试的其它9种水产常见病原菌PCR扩增均呈阴性；能够检测的最低细菌数在20～30个细菌；方法可直接从病鱼的脑、肝脏、肾脏及脾脏组织检测到该菌；另外，临床菌株检测结果与基于菌株16S rRNA基因序列系统进化分析结果一致。该方法弥补了传统细菌鉴定很难将该菌鉴定到种的缺点，并显著缩短了检测时间及降低了检测成本，具有较好的应用前景。

In recent years, Streptococcus iniae infected farmed tilapia causes economic losses in our country and the world. To set up a method detecting S. iniae in tilapia, this paper designed primers CM1/CM2 based on the sequences of S. iniae. At the same time, the tests which amplified the specific DNA fragment, optimized the PCR reaction condition, sensitiveness and special were detected. The different detected material was compared and 9 clinical samples were detected at one time. The result indicated that the CM1/CM2 primers set only amplified a specific DNA fragment from S. iniae but not from 9 strains common pathology bacteria of fisheries. It can detect bacterial cells in 20 -30, and the PCR reaction also can directly detect the S. iniae from brain, liver, kidney, spleen of infected tilapia. On the other hand, the detection results of clinical strains were consistent with the Phylogenist Analysis based on 16S rRNA gene sequences. The method makes up for the disadvantage of traditional method that can not detect the species between the bacteria and reduce the detected time and the cost. So, the method detecting S. iniae will have a good future for application.