使用16SrDNA序列分析鉴定副溶血性弧菌

Identification of Vibrio parahaemolyticus from Other Clinical Isolated Bacteria by Partial 16SrDNA Sequencing

目的 测试应用16SrDNA序列分析鉴定副溶血性弧菌的适用性。方法用16SrDNA基因的扩增引物，对从腹泻者，水产品和环境中分离获得的副溶血性弧菌（n=39）及临床分离的15种（n=181）常见菌的基因组DNA进行聚合酶链反应，产物经琼脂糖凝胶电泳纯化后直接测序。序列通过blastn，在GenBank中比对，确定细菌属种并与表型法结果进行比较。结果220株菌（220／220）扩增出870bp的目的片段，平均测序长度752bp。36／39株副溶血性弧菌鉴定结果与生化培养法相符，3株菌株分别鉴定为科氏葡萄球菌（Staph3，lococcuscohnii），表皮葡萄球菌（Staphylococcusepidermidis）和Oceanobacillus profundus。181株其它临床分离菌无误判为副溶血性弧菌，其中123株与生化培养法结果一致，58株有差异。结论本文报告的16SrDNA基因序列分析可用于准确鉴定副溶血性弧菌，并可与15种临床常见分离菌区分。

Objective To evaluate the feasibility to employ 16S rDNA sequencing as a test for Vibrio parahaemolyticus identification. Methods Vibrio parahaemolyticus strains （n=39） were individually isolated from diarrhea patients, fishery products and environment water samples respectively. Other fifteen species of clinical bacteria isolates （n=181） were included as controls. Bacteria genome DNAs were amplified by PCR with primers designed for partial 16S rDNA. Then, amplified products were purified from agarose-gel electrophoresis, directly Sequenced by BigDye 3.0 kit. Sequences were compared with database in GenBank using program blastn for the identification of the bacterial species. Results were further compared with those identified by routine biochemistry tests. Results All of 220 bacteria isolates were successfully amplified by PCR with a 870 bp target fragment. The 16S rDNA test can identify thirty-six out of 39 Vibrio parahaemoly- ticus isolates （92.30%）, in accordance with the results determined by routine biochemistry test. None of 181 other clinical bacterial isolates was classified as Vibrio parahaemolyticus by 16S rDNA test, although 41 of them were not consistant with the results from biochemistry test. Conclusions These data suggested that 16S rDNA test can precisely identify Vibrio parahaemolyticus from most of common clinical isolates.