Detection of Pathogenic Bacteria from Fishes and Their Environments by PCR

(1)根据16 SrRNA序列设计了嗜冷噬纤维菌、柱状噬纤维菌、海洋嗜热杆菌和嗜鳃黄杆菌的特异性PCR引物。(2)1995年对琵琶湖香鱼鱼苗和银鲑卵进行了嗜冷隐孢子虫带菌检测，结果显示，35%（7/20）的成鱼和12%（18/146）的成鱼为阳性。1996年检出率分别为80%（4/6）和33%（6/18）。在养殖苗种用银鲑卵中，美国野生种的7批中有5批为阳性，美国和日本家种的4批均为阴性。(3)1996年在广岛县的一个私人养殖场采集了10只虾类样品，用RT-PCR检测对虾弧菌，其灵敏度是16 SrDNA的100倍。在100只对虾中，有10只对虾的RT-PCR检测结果为阳性。(4)建立了一种基于16 SrRNA的杀鱼巴氏杆菌特异性RT-PCR方法。在爱媛县健康海水鱼中PCR检测到P.piscida，养殖和野生黄尾鱼均处于高携带状态。(5)根据从杀鱼潜蝇特异性质粒pzP 1中克隆的DNA片段的核苷酸序列，构建了两对引物。在自然感染的黄尾鱼肾脏DNA中检测到PCR产物，而在健康黄尾鱼肾脏DNA中未检测到PCR产物。(6)一种杀鲑气单胞菌亚种特异性DNA片段。从RAPD产物中克隆出杀鲑素基因。基于克隆的核苷酸序列，合成PCR引物组。该方法能有效检测杀鲑游动放线菌。实验感染的鲑鱼肾脏中的杀鲑素。

(1) PCR primers specific for Cytophaga psychrophila, Cytophaga columnaris, Flexibacter maritimus, and Flavobacteium branchiophilum, were constraucted on the basis of the 16S rRNA sequence.(2) Carrier detection of C.psychrophila in ayu fry and coho salmon eggs was made by nested-PCR.In ayu sampled from the Biwa Lake, 35 percent (7/20) of the fish lots and 12 percent of the total fish examined (18/146) were positive in 1995, and 80 percent (4/6) of the fish lots and 33 percent (6/18) of the total fish were positive in 1996. In coho salmon eggs for aquaculture seedlings, five of seven lots from the wild spawner in USA were positive, and all four lots of eggs from domestic spawners in USA and Japan were negative.(3) For detection of Vibrio penaecida 16S rRNA-targeted RT-PCR was demonstrated to have 100 times higher sensitivity than 16S rDNA targeted PCR.Ten kuruma prawn for each sampling were collected at a private farm in Hiroshima Prefecter in 1996. Out of 100 prawn examined, ten prawn were positive in RT-PCR.(4) A 16S rRNA based RT-PCR method specific for Pasteurella piscicida was developed. The PCR detection of P.piscida in apparently healthy marine fishes in Ehime Prefecture had a high carrier state in both farmed and wild yellowtail.(5) Based on the nucleotide sequences of DNA fragments cloned from a plasmid pzP1 specific to P.piscicida, two primer sets were constructed. The PCR products were detected in DNA from the kidney of yellowtail infected naturally with P.piscicida, but not in that from healthy yellowtail.(6) A DNA fragment specific to Aerkomonas salmonicida subsp. salmonicida was cloned from the RAPD products. Based on the nucleotide sequence of the clone a PCR primer set was synthesized. It was effective in detecting A.salmonicida subsp. salmonicida from the kidney of experimentally infected amago salmon.