矛尾复鰕虎鱼病原哈氏弧菌的鉴定及特异性检测方法的建立

取体表出血、肌肉溃疡的虾蟹养殖塘混养大量死亡的矛尾复鰕虎鱼的深层溃烂组织及肝脏进行细菌分离,2种被检组织中均分离到2种优势生长菌落;人工感染试验表明,其中的一株分离菌(S090801)浓度为106～108 cfu/ml可引起矛尾复鰕虎鱼全部死亡,该菌的形态特征、生理生化特性及基于16SrRNA和gyrB 2种基因的系统发育学分析确定为弧菌属的哈氏弧菌。同时基于gyrB基因序列设计1对特异性引物,建立了一种哈氏弧菌快速、敏感的PCR检测方法,扩增目的片段大小为363bp,该方法检测哈氏弧菌模板DNA最低质量浓度为0.046875ng/μl,可用于哈氏弧菌引起的水产动物疾病的快速诊断及分子流行病学的调查研究。

Identification and PCR Detection of Pathogenic Vibrio harveyi Isolated from Gobby Synechogobius hasta

The outbreaks of mass mortality of pond-cultured gobby Synechogobius hasta occurred in some ponds in Ganyu,Jiangsu province from August to September in 2009.Two dominant bacteria were isolated from liver and ulcer tissues in the gobby with haemorrhages at the body surface,ulcer of muscle,and all inoculated to the fish,leading to died,post-injection with 0.1 ml live cells(106~108cfu/ml,S090801) per fish.The morphological,physiological and biochemical characteristics,and phylogeney analysis based the 16S rRNA and gyrB genes showed that the pathogen in the diseased gobby was considered to be a bacterium Vibrio harveyi.In addition,based on the gyrB gene sequences,a pair of specific primers was designed,and then a rapid and accurate polymerase chain reaction(PCR) detecting V.harveyi were established.The PCR amplified 363 bp gene fragment from chromosomal DNA of V.harveyi,and detect purified chromosomal DNA at a level as low as 0.046875 ng/μl.The PCR protocol is suggested to be used in the specific and rapid diagnose and epidemiological investigation of the disease caused by V.harveyi.