刺参腐皮综合征重要病原灿烂弧菌DNA的探针制备及应用

腐皮综合征是近年来刺参养殖最重要的疾病，导致刺参大批死亡和惨重的经济损失。以其主要致病原灿烂弧菌（Vibrio splendidus）DNA为模板，PCR扩增出16s-23s间隔区序列，将该片段克隆进pMD19-T Vector，转化大肠杆菌DH5a，获得重组质粒转化菌并进行测序。根据序列、设计引物，合成177bp的地高辛标记DNA探针。该探针对灿烂弧菌的DNA呈现特异性，而对其它细菌Vibrio fluvialis、Vibrio anguillarum、Vibrio alginolyticus、Aeromonas hydrophila、Vibrio harveyi、Vibrio parahaemolyticus、Vibrio vulnificus的核酸均呈阴性；探针对灿烂弧菌DNA的检出极限为6.25pg, 具有较高敏感度。应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探针检测，其检出率均为100%。研究结果表明，该探针具有良好的特异性和敏感性，可成功用于快速检测养殖刺参“腐皮综合征”重要病原灿烂弧菌。该方法应用于刺参腐皮综合征的检测尚属首次，它将为刺参腐皮综合征的快速诊断、疾病防治和养殖生产提供技术支撑和参考。

Application and Establishment of DNA Probe for the Pathogen Vibrio splendidus Causing Skin Ulcer Syndrome in Apostichopus japonicus

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 177bp) 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 6.25pg 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.