Institute for Coastal Marine Environment, CNR, Spianata S. Raineri 86, 98122 Messina, Italy.
Res Microbiol. 2011 Apr;162(3):223-30. doi: 10.1016/j.resmic.2010.11.004. Epub 2010 Dec 8.
Vibrio anguillarum is a pathogen that causes high mortality in marine and freshwater fish. The aim of this study was to develop a real-time PCR assay for identification and quantification of V. anguillarum in fish tissue. The assay was carried out using two target genes, 16SrDNA and toxR, to evaluate the influence of differences in the operon copy number in quantitative assessment, both in pure cultures of V. anguillarum serovar O1 (strain 975/I), as a reference, and in the liver and kidney of a sea bass (Dicentrarchus labrax) specimen. Real-time PCR analysis showed high specificity for both target genes, with a detection limit of approximately 1-10 bacterial cells per reaction in pure culture and 10/100 V. anguillarum cells per reaction in fish tissue, which corresponds to 2 × 10(2)/2 × 10(3) cells g(-1) fish tissue. Moreover, both genes showed high specificity but differing sensitivity due to the different operon copy number; as a result, it is possible to target the high copy number gene to improve sensitivity. Our results suggest that the protocol we tested can be used as a sensitive and specific molecular method for the detection of the fish pathogen V. anguillarum in fish tissue.
鳗弧菌是一种引起海水和淡水鱼类高死亡率的病原体。本研究旨在开发一种用于鉴定和定量检测鱼类组织中鳗弧菌的实时 PCR 检测方法。该检测方法使用 16SrDNA 和 toxR 两个靶基因，以评估在定量评估中，在鳗弧菌血清型 O1（菌株 975/I）的纯培养物以及鲈鱼（Dicentrarchus labrax）肝脏和肾脏标本中，操纵子拷贝数差异对其的影响。实时 PCR 分析显示两种靶基因都具有很高的特异性，在纯培养物中，每个反应的检测限约为 1-10 个细菌细胞，在鱼类组织中，每个反应的检测限为 10/100 个鳗弧菌细胞，相当于 2×10(2)/2×10(3)个细胞/g 鱼组织。此外，由于操纵子拷贝数的不同，两种基因都具有高特异性但敏感性不同；因此，可以针对高拷贝数基因来提高灵敏度。我们的结果表明，我们测试的方案可以作为一种敏感且特异性的分子方法，用于检测鱼类组织中的鱼类病原体鳗弧菌。
