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从山东沿海的发病鲈鱼(Lateolabrax japonicus)分离到1株致病性哈维氏弧菌(Vibrio harveyi),从该菌的染色体DNA扩增出一条长约1.4 kb的特异性片段。DNA序列分析表明,该克隆片段含有完整的1 254 bp溶血素基因,该溶血素基因与哈维氏弧菌VIB645的溶血素基因VhhA和VhhB的相似性分别为99.0%和98.5%;与副溶血弧菌(V.parahaemolyticus)热稳定性溶血素基因(TDH)的相似性为74.5%;与拟态弧菌(V.mimicus)、创伤弧菌(V.vulnificus)、霍乱弧菌(V.cholerae)磷脂酶基因的序列相似性分别为57.4%、59.2%、53.0%;与最小弧菌、创伤弧菌、霍乱弧菌、霍氏弧菌(V.hollisae)、河流弧菌(V.fluvialis)、鳗弧菌(V.anguillarum)的溶血素基因的相似性仅为19.9%~24.8%。根据溶血素基因的保守区段,设计了1对特异引物。分析表明,该引物能特异检测哈维氏弧菌,其可检测的DNA最小量为0.001 ng。用该方法对55株不同来源的患病鱼分离的疑似病原菌进行检测,结果检出8株哈维氏弧菌,检出率为14.55%,从健康动物中检出数占所检弧菌数的6.25%,海洋环境为10.53%,海水养殖环境为26.53%,表明哈维氏弧菌在不同的海水环境及健康海洋动物中普遍存在,在养殖水体中的数量高于其他海水环境。
One pathogenic Vibrio harveyi was isolated from the seabed sea bass (Lateolabrax japonicus) in Shandong Province and a 1.4 kb specific fragment was amplified from the chromosomal DNA of the strain. DNA sequence analysis showed that the cloned fragment contained a complete 1 254 bp hemolysin gene whose homologies with the VhhA and VhhB genes of V. harveyi VIB645 were 99.0% and 98.5%, respectively. The similarity of V. parahaemolyticus thermostable hemolysin gene (TDH) was 74.5%; the similarity with V. mimicus, V. vulnificus, V. cholerae, The sequence similarity of the phospholipase gene was 57.4%, 59.2% and 53.0%, respectively. The results showed that the sequence similarity of the phospholipase gene to Vibrio mimicus, Vibrio vulnificus, V. cholerae, V. hollisae, V. fluvialis, The similarity of the hemolysin genes of V. anguillarum was only 19.9% ~ 24.8%. According to the conserved region of hemolysin gene, one pair of specific primers was designed. The analysis showed that this primer could specifically detect Vibrio harveyi and the minimum detectable DNA was 0.001 ng. The method was used to detect the suspected pathogenic bacteria isolated from 55 diseased fish from different origins. As a result, 8 strains of Vibrio harveyi were detected, the detection rate was 14.55%. The number of Vibrio parahaemolyticus , 6.25% in the marine environment, 10.53% in the marine environment, and 26.53% in the marine aquaculture environment. This indicates that Vibrio harveyi is ubiquitous in different marine environments and healthy marine animals, and is higher in aquaculture waters than in other seawater environments.