16S rDNA克隆文库法分析生鲜牛乳中 细菌种群的多样性

Study on bacterial population diversity in raw milk with 16S rDNA library analysis method

中文关键词:  16S rDNA  克隆文库  生鲜牛乳  种群多样性  致病菌

英文关键词:16S rDNA  clone library  fresh milk  population diversity  pathogenic bacteria

中文摘要:

      目的 利用16S rDNA基因文库分析法, 对生鲜牛乳中微生物的种群多样性进行研究。方法 提取生鲜牛乳中总微生物基因组DNA为模板, 将扩增到的生鲜牛乳样品的16S rDNA的PCR产物与pMD~18T载体连接, 构建其菌群的16S rDNA文库。对随机选取的56个阳性克隆子进行序列测定和BLAST比对。结果 文库序列分析表明, 有53个克隆子分属3个不同的类群, 即厚壁菌类群、变形菌类群和异常球菌-栖热菌类群, 其余3个克隆子属于未知类群。其中, 优势细菌类群为厚壁菌类群(86%), 在该类群中, 尤以无乳链球菌为主要代表, 约占所有克隆子的82%; 其他类群为γ~变形菌类群(7.1%)和异常球菌-栖热菌门类群(1.8%)。系统发育分析表明, 未知类群在分类地位上更接近与厚壁菌类群。结论 生鲜牛乳中微生物具有多样性, 无乳链球菌为其中的优势种群, 同时生鲜牛乳中还存在葡萄球菌等致病菌, 或不动杆菌和肠道菌等条件致病菌。

英文摘要:

      Objective Using 16S rDNA gene clone library analysis method, the bacterial population diversity in raw milk was analyzed. Methods Microbial genomic DNA in raw milk was extracted and used as template to analysis．PCR products of 16S rDNA in raw milk were ligated with vector pMD~18T, constructing a bacterial gene clone library. 56 positive clones were picked randomly from the 16S rDNA library, followed by sequence analysis and BLAST comparison. Results Sequence analysis showed that 53 clones could be di-vided into 3 groups: Firmicutes , Proteobacteria , and Deinococcus~Thermus. The rest of 3 clones were uncultured bacteria. The dominant group belonged to Firmicutes (86%), in which Streptococcus agalactiae predominated, and possessed 82% of all clone in the library. Other groups were γ~Proteobacteria (7.1%) and Deinococcus~Thermus (1.8%). And the uncultured bacteria were closed to Firmicutes in the phylogenetic tree. Conclusion The bacterial community composition in raw milk was of high diversity. Streptococcus agalactiae was dominant population while other pathogenic bacteria, such as Staphylococcus or conditioned pathogen, like Acinetobacter sp. and Enterobacter sp. were also existed in raw milk.