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作 者:张凤萍[1,2,3] 王印庚[1] 李胜忠[3] 荣小军[1] 廖梅杰[1] 张正[1]
机构地区:[1]农业部海洋渔业资源可持续利用重点开放实验室中国水产科学研究院黄海水产研究所,青岛266071 [2]浙江舟山市水产研究所,316000 [3]新疆农业大学动物科学学院,乌鲁木齐830052
出 处:《海洋水产研究》2008年第5期100-106,共7页Marine Fisheries Research
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);山东省农业良种工程课题;农业部农业结构调整重大技术研究专项(06-05-04B)资助
摘 要:腐皮综合征是近年来刺参养殖最主要的疾病,导致刺参大批死亡和惨重的经济损失。根据其主要致病原灿烂弧菌(Vibrio splendidus)的16S^23S间隔区序列,设计特异性引物,利用PCR技术,从纯化的V.splendidus DNA中成功地扩增出产物大小为177bp的DNA片断,并对此方法的灵敏度和特异性进行了研究。结果表明,PCR技术对V.splendidus DNA的检测灵敏度为0.5pg,并且对灿烂弧菌的DNA呈现特异性,而与其他细菌V.fluvialis、V.anguillarum、V.alginolyticus、Aeromonas hydrophila、V.harveyi、V.parahaemolyticus、V.vulnificus的DNA均无交叉反应。应用PCR技术对人工感染的以及取自青岛、烟台和威海的腐皮综合征发病海参进行检测,其检出率均为100%。研究结果表明,PCR方法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原灿烂弧菌。在国内,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及防治提供又一新的方法。The skin ulcerative syndrome is a serious disease causing massive mortality and economic losses in cultured sea cucumber Apostichopus japonicus in recent years. The PCR primers for the detection of V. splendidus were designed according to the sequence of the 16 5-23 S rDNA intergenic spacers. In this study, the technology of PCR was developed and applied to amplify a 177bp DNA fragment from the DNA of purified V. splendidus. And the sensitivity and specificity of this PCR method were studied. The sensitivity test showed that the detection limit of PCR was 0. 5pg V. splendidus DNA, and it was only positive for V. splendidus DNA, and negative for DNAs of V. fluvialis, V. anguillarurn, V. alginolyticus, V. parahaemolyticus, V. harveyi, V. vulnificus and Aeromonas hydrophila. In addition, this method was considered to be accurate in the detection of V. splendidus as the positive ratio was as high as 100% for the tissue samples of clinically infected A. japonicus and the samples showing ulcerative syndrome from Qingdao, Weihai and Yantai areas. This method was established and applied in China to detect V. splendidus in A. japonicus. As it is sensitive and accurate in detection of the virus, it would be helpful in disease control and health management of cultured A japonicus.
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