RT-PCRによるブリからのPhotobacterium damselaの検出

魚類病原細菌 Photobacterium damsela subsp. damsela および Ph. damsela subsp. piscicida を検出するために、本菌の 16S rRNA における種特異的塩基配列の解析に基づきRT-PCR法を考案した。まず、魚介類病原菌を中心とした12種のビブリオ属細菌を用いて本RT-PCRの特異性を確認し、また検査魚の脾臓磨砕液をトリプトソーヤ・ブロスで前培養後RT-PCRに供することにより検出感度が高まることが分かった。この方法を用いることにより、外見的に健康な養殖ブリ Seriola quinqueradiata およびウマヅラハギ Thamnaconus modestus やマアジ Trachurus japonicus など数種の天然魚に Ph. damsela が存在することが明らかにされた。

An RT-PCR detection technique was developed for Pholobacterium damsela (=Photobacterium damsela subsp. damsela and Ph. damsela subsp. piscicida) from apparently healthy yellowtail (Seriola quinqueradiata) and other wild fishes, based on the analysis of a species-specific sequence in 16S rRNA of Ph. damsela. Specificity of the RT-PCR with K-F1 (sense primer) and R-GEN (antisense primer) was confirmed by using Ph. damsela subsp. damsela and Ph. damsela subsp. piscicida, 12 strains of fish—associated Vibrio species. Sensitivity of the RT-PCR was improved by combining a pre-culture of the sample (spleen homogenate) with tryptic soy broth (NaCl 2%). This RT-PCR revealed the presence of Ph. damsela from wild yellowtail, filefish (Thamnaconus modestus) and horse mackerel (Trachurus japonicas) caught in the sea as well as apparently healthy cultured yellowtail.