刺参腐皮综合征病原灿烂弧菌检测探针的制备及应用  

Detection of Vibrio splendidus causing skin ulcer symdrome of sea cucumber(Apostichopus japonicus) with DNA probes

作　　者：王印庚[1] 张凤萍[1,2] 李胜忠[3] 陈霞 崔玉龙[5] 

机构地区：[1]中国水产科学研究院黄海水产研究所海水鱼类养殖与设施渔业研究室,山东青岛266071 [2]浙江省舟山市水产研究所,浙江舟山316000 [3]新疆农业大学动物科学学院,新疆乌鲁木齐830052 [4]青岛九洋红水产科技有限公司,山东青岛266071 [5]荣成市海洋与渔业局,山东威海264309

出　　处：《水产学报》2009年第1期119-125,共7页Journal of Fisheries of China

基　　金：山东省科技发展计划项目(2004GG2205116);青岛科技发展计划项目(02-1-kchhh-44);农业部农业结构调整重大技术研究专项(06-05-04B);山东省农业良种工程重大课题

摘　　要：腐皮综合征是近年来养殖刺参发生的严重疾病。发病时,刺参大批死亡,经济损失惨重。以其主要致病原灿烂弧菌(Vibrio splendidus)DNA为模板,PCR扩增出16S-23S核糖体RNA基因间隔区序列,插入pMD19-T,转化大肠杆菌DH5α并测序。根据该间隔区序列,设计引物扩增177 bp的地高辛标记的探针。该探针对灿烂弧菌呈现特异性,而对其它细菌V.fluvialis,V.anguillarum,V.alginolyticus,Aeromonas hydrophila,V.harveyi,V.parahaemolyticus,V.vulnificus均呈阴性。探针对灿烂弧菌DNA的检出限为6.25 pg,具有较高敏感度。用该探针对人工感染以及从青岛、烟台、威海获取的腐皮综合征发病海参和养殖水体进行斑点杂交,灿烂弧菌检出率达100%。结果表明,该探针具有良好的特异性和敏感性,可用于快速检测养殖刺参腐皮综合征的病原灿烂弧菌。用该方法检测刺参腐皮综合征尚属首次,为刺参腐皮综合征的快速诊断和防治提供技术支撑。The skin ulcer syndrome is a serious disease causing massive mortality and economic losses in cultured sea cucumber （Apostichopus japonicus）. The causative agent was identified as Vibrio splendidus. As V. splendidus DNA was gained, the 16S -23S rDNA intergenic spacers were amplified by PCR, and cloned into pMD19-T vectors and finally sequenced. A fragment （about 177 bp） was obtained by PCR, and it was labeled with digoxigenin-dNTP to form a dot-blot hybridization probe for V. splendidus DNA. The results showed that the probe was only positive for V. splendidus DNA, while it was all negative for DNAs of bacteria V. fluvialis, V. anguillarum, V. alginolyticus, V. parahaemolyticus, V. harveyi, V. vulnificus and Aeromonas hydrophila. The sensitivity test illustrated that the detection limit of the dot-blot hybridization probe was 6.25 pg V. splendidus DNA. In addition, the probe was considered to be accurate in the detection of V. splendidus as the positive ratio was as high as 100% for the total samples from Qingdao, Weihai and Yantai areas. This probe was the first time to be established and applied in China to detect V. splendidus in A. japonicus. As it is sensitive and accurate in detection of V. splendidus, it would be helpful in the disease control and health management in cultured A. japonicus.

关 键 词：刺参 腐皮综合征 灿烂弧菌 聚合酶链式反应 斑点杂交 

分 类 号：S941.42[农业科学—水产养殖]