养殖刺参“腐皮综合征”致病菌--灿烂弧菌的原位杂交检测方法的建立与应用

Establishment and application of in situ hybridization detection of Vibrio splendidus causing skin ulcer syndrome in cultured Apostichopus japonicus

以养殖刺参"腐皮综合征"的重要致病菌——灿烂弧菌Vibrio splendidus的16S～23S间隔区序列,设计特异性引物,用PCR方法制备地高辛(DIG)标记探针,建立了原位杂交技术检测感染刺参体内灿烂弧菌的技术方法,并利用该方法对人工感染刺参和健康刺参各组织进行检测。结果显示,感染刺参的体壁结缔组织、肌肉组织、肠粘膜上皮、辐水管等组织的原位杂交检测呈阳性,而与健康刺参组织无交叉反应。在感染组织中,阳性信号(显色)强弱清晰,能准确反映出灿烂弧菌的侵染部位及感染程度,这为探明灿烂弧菌的感染途径、感染病程等致病机理研究奠定了基础,也为养殖刺参疾病预防和健康管理提供了技术支撑。

The skin ulcer syndrome is a serious disease causing massive mortality and eco- nomic losses in cultured sea cucumber Apostichopus japonicus. Vibrio splendidus was identi- fied as one of the most severely pathogeneses of this disease. In the present study, a DIG-la- beled V. splendidus DNA probe based on the sequence of the 16S- 23S rDNA intragenic spac- ers was synthesized and was used to establish in situ hybridization for detecting the bacterium inA. japonicus. V. splendidus-positive cells were observed in connective tissue, muscle tissue, epithelium of intestine and water-vacular tissues in artificial infected A. japonicus, while no cross-reaction was observed in tissues of healthy sea cucumber. The positive signals were clear in infected tissues, which could reveal the location and degree of the infection. Therefore, this established approach is useful to indicate the infection route and the pathogenesis of V. splendi- dus infection, which will provide basic data for disease control and animal health management.