作　　者： ; ; ;

机构地区： 广东海洋大学水产学院广东省水产经济动物病原生物学及流行病学重点实验室

出　　处： 《华中农业大学学报》 2009年第4期459-462,共4页

摘　　要： 从基因库中获取溶藻弧菌的胶原蛋白酶基因序列和外膜蛋白基因序列,根据基因的保守性用DNAStar分别设计2对引物,分别对溶藻弧菌基因组进行PCR扩增,用地高辛标记扩增的特异DNA片段以制备探针。分别用胶原蛋白酶DNA探针和外膜蛋白DNA探针对实验室保种的溶藻弧菌以及鱼排分离的溶藻弧菌进行斑点杂交检测,检测结果表明,胶原蛋白酶基因探针与保种的溶藻弧菌以及鱼排分离的溶藻弧菌DNA产生较强的阳性反应,而与霍乱弧菌、哈氏弧菌和副溶血弧菌DNA等均无反应,表明该胶原蛋白酶DNA探针的特异性较强;而外膜蛋白基因探针与保种的溶藻弧菌以及鱼排分离的溶藻弧菌DNA产生较强的阳性反应,但与哈氏弧菌和副溶血弧菌DNA也出现了较强的阳性反应,与霍乱弧菌DNA则无反应,表明该外膜蛋白DNA探针的特异性较低。说明所构建的溶藻弧菌胶原蛋白酶DNA探针斑点杂交检测方法具有特异性强,简便易行,可应用于溶藻弧菌的快速检测。 Two pairs of sequences of the collagenase primers were designed using DNAStar software based on the conservative and outer membrane gene of Vibrio alginolyticus in the GenBank. These two pairs of specific primers were applied to amplify V. alginolyticus genomic DNA by PCR, and the two PCR products were labeled by using digoxigenin to prepare the collagenase DNA probe and outer membrane protein DNA probe. The dot blotting detection method was established to detect the V. alginolyticus and other vibrios using the digoxigenin-labeled probes. The results showed that the collagenase DNA probe could positively react with all V. alginolyticus, but not with V. cholerae,V, harveyi and V. parahaemolyticus. The outer membrane protein probe could positively react with V. alginolyticus, but also positively with V. harveyi and V. parahaemolyticus, while negatively with V. cholerae. Above results suggested that the collagenase DNA probe had higher specificity to detect V. alginolyticus than the outer membrane protein probe. Therefore;the established dot-blotting detection method via V. algino- lyticus collagenase DNA probe is characteristic by simplicity, higher specificity and possibly applied for the rapid detection of V. alginolyticus.

关 键 词： 溶藻弧菌 胶原蛋白酶 探针 外膜蛋白 探针 斑点杂交

领　　域： [生物学]