一株羊源单核细胞增生性李斯特菌的

一株羊源单核细胞增生性李斯特菌的 一株羊源单核细胞增生性李斯特菌的分离与鉴定 马光强1，刘丽娟1，文 明1,2，王开功1,2，程振涛1,2*，周碧君1,2* （1. 贵州大学动物科学学院，贵州 贵阳 550025, 2.贵州省动物疫病与兽医公共卫生重点实 验室，贵州 贵阳 550025） 摘要:本研究旨在了解一株单核细胞增生性李斯特菌（Lm）贵州分离株的理化特性及遗传变异情况，应用实验室细菌分离培养技术从贵州省盘县某养羊场疑似病例组织样本中分离该单核细胞增生性李斯特菌，并对该菌进行鉴定。合成Lm的hly基因引物，以分离菌株基因组DNA为模板，扩增hly基因，目的基因经回收纯化与pMD19-T载体连接后转化感受态细胞DH5α，重组质粒经PCR、双酶切鉴定后进行测序及序列。结果显示从临床疑似山羊病例中分离到培养特性、菌落形态、菌体形状特征、生理生化特征均与文献报道相同的疑似单核细胞增生性李斯特菌。从该分离菌株基因组中扩增到约850bp的Lm的特异性DNA片段，其序列与GenBank数据库中其他Lm地方株核苷酸同源性在39.1%-99.7%之间，其中与从发病动物分离到的加拿大、瑞士参考株同源性最高，均达到99.7%，与其他途径分离到的参考菌株同源性都很低，这从分子水平上进一步证实分离菌株为单核细胞增生性李斯特菌，且从不同途径分离的地方株hly基因差异大。研究结果为本病临床防控与治疗提供理论数据。 关键词:单核细胞增生性李斯特菌；分离鉴定；hly基因；序列分析 Isolation and Identification of a Listeria monocytogenes in Caprine MA Guang-qiang1, LIU Li-juan1,2,WEN Ming1,2,WANG Kai-gong1,2,CHENG Zhen-tao1,2*, ZHOU Bi-jun1,2* （1. College of Animal Science, Guizhou Uiversity, Guiyang 550025, China; 2. Key laboratory of animal disease and veterinary public health in guizhou province, Guiyang 550025, China） Abstract: This study aims to understand the characterization and genetic variation of a Listeria monocytogenes (Lm) in Guizhou, applying the laboratory isolation and culture techniques isolated a Lm from the tissue samples of a infected case in a Caprine farm in Pan County of Guizhou，and use the traditional test methods for its identification. A pair of specific primer designed and synthesized to amplify the sequence of hly gene in isolated Lm strain’s genomic DNA. The hly was cloned into pMD19-T vector, positive colonies were sequenced and digested by restriction endonuclease after transforming, Sequence analysis for it then. The results showed that a suspected Lm have been isolated from a suspected Caprine case, and its cultural characteristics, colony morphology, shape features, and Physiological-biochemical characteristics are the same as the Lm in literature reports. A specific DNA fragment about 850bp of Lm was amplified, and the nucleotide homology between 39.1% -99.7% compared with part of local strains in GenBank, Canada and Switzerland（99.7% ）isolated in onset of animals is the highest, very low with the other strain isolated from other ways, which is further confirms from the 基金项目：贵州省农业攻关项目“畜禽源主要病原菌药物防控技术创新与示范”（黔科合NY[2014]3042号）。 作者简介：马光强(1987-)，男，贵州石阡人，在读硕士，主要从事动物疫病细菌学研究。 *通讯作者，Tel: 0851-88298078, Email: chengzhentao@sohu.com；as.bjzhou@gzu.edu.cn。