半滑舌鳎病原鳗利斯顿氏菌双重PCR检测方法的研究  

Detection of pathogenic Listonella anguillarum isolated from Cynoglossus semilaevis by duplex PCR

作　　者：张晓君[1] 阎斌伦[1] 梁利国[1] 秦国民[1] 毕可然[1] 

机构地区：[1]淮海工学院海洋学院江苏省海洋生物技术重点建设实验室,江苏连云港222005

出　　处：《海洋通报》2011年第4期430-434,共5页Marine Science Bulletin

基　　金：江苏省水产三项工程项目(PJ2010-58);江苏省自然科学基金项目(BK2009163)

摘　　要：对引起江苏连云港工厂化养殖半滑舌鳎大量死亡的病原鳗利斯顿氏菌,进行了基于溶血素和金属蛋白酶两种基因的双重PCR检测。根据鳗利斯顿氏菌溶血素基因和金属蛋白酶基因序列设计2对特异性引物,在同一PCR反应体系中,鳗利斯顿氏菌可同时扩增出上述2种基因片段,扩增片段大小分别为493 bp和248 bp,两对引物对5种其他水产动物病原菌无交叉反应,敏感性检测结果为该双重PCR最低能检测8×102 CFU/mL的鳗利斯顿氏菌,最低能检测0.171 875 ng/μL的鳗利斯顿氏菌基因组DNA。对发病半滑舌鳎溃疡组织、肝脏及养殖用水进行双重PCR检测,呈阳性反应的样品可分离出优势生长的鳗利斯顿氏菌。该实验表明两种不同基因综合检测病原鳗利斯顿氏菌,进一步提高了其PCR检测的准确性和特异性,建立了一种病原鳗利斯顿氏菌快速、准确的双重PCR检测方法。L.anguillarum is the cause of the high mortalities of cultured half-smooth tongue sole（Cynoglossus semilaevis） during cultivation in Lian Yungang,Jiangsu province.In this study,the detection of the pathogenic L.anguillarum by duplex PCR based on hemolysin and metalloprotease genes were conducted.Two pairs of specific primers were designed based on hemolysin and metalloprotease gene sequence,and these two primers could simultaneously amplify 493 bp and 248 bp gene fragments from chromosomal DNA of L.anguillarum in one PCR reaction,and no cross reaction was detected in 5 other pathogenic bacteria tested.The results of sensitivity of dulplex PCR showed that the two primers could detect L.anguillarum as low as 8×102CFU/mL,and detect purified chromosomal DNA as low as 0.171 875 ng/μL.Dominant L.anguillarum could be isolated from the positive sample of duplex PCR through detecting ulcer tissues and liver of diseased Cynoglossus semilaevis.The specific PCR method for detection of L.anguillarum was established,and the PCR protocol simultaneously amplifying different gene fragments of the L.anguillarum could be useful in the specific,accurate and rapid detection of pathogenic L.anguillarum.

关 键 词：半滑舌鳎 鳗利斯顿氏菌 溶血素基因 金属蛋白酶基因 双重PCR 

分 类 号：S917.1[农业科学—水产科学]