81株铜绿假单胞菌16SrRNA基因序列测定及系统发育学分析

摘要

；采用16S rRNA 基因序列分析法对2015年7~9月广东省食品检验所桶装水专项抽检中筛查所得的81株铜绿假单胞菌(Pseudomonas aeruginosa )进行复核鉴定。菌株经纯化培养后提取总 DNA,采用细菌16S rRNA 通用引物进行16S rRNA 基因序列扩增,PCR 扩增产物经2%琼脂糖凝胶电泳后,进行序列测定,序列经人工校对后用 Clustal X 进行比对分析,最后用 MEGA5.1软件构建系统发育树。系统发育分析结果表明：81株铜绿假单胞菌与原鉴定结果一致。其中,编号24-3-QY、100-5-JM、106-3-JM 菌株形成一个分支,28-1-WD 单独为一支,其余77株野生菌和铜绿假单胞菌标准菌株 ATCC27853聚为一群。该研究是2015年5月24日中国开始实施 GB 19298—2014《食品安全国家标准包装饮用水》以来,广东省首次对水源性铜绿假单胞菌进行的研究,为下一步菌种污染朔源等研究提供依据。%81 P seudomonas aeruginosa from Guangdong Provincial Institute of Food Inspection were identified by 1 6S rRNA sequence a-nalysis.The DNA was isolated and the sequences of 1 6S rRNA gene were amplified by PCR with the bacterium universal primers,and then the PCR products were sequenced after 2% agarose gel electro-phoresis.Moreover, the corrected sequences were aligned with Clustal X and the phylogenetic tree was constructed by MEGA5.1 . Consequently,the identified results of the 81 strains confirmed their original identification before.On the phylogenetic tree,No.24-3-QY strain formed a separate branch with No.100-5-JM strain and No. 106-3-JM strain.No.2 8-1-DW formed one branch and the other 77 strains formed a separate branch with P .aeruginosa ATCC 27853. This was the first research about waterborne P .aeruginosa in Guangdong Province when China began to implement GB 1 9298—2014 “National food safety standards of packaged drinking water”since May 24, 2015.The waterborne P .aeruginosa culture collection in Guangdong was preliminarily established basing on these strains.These data will provide a powerful tool for effectively tracing source of P . aeruginosa and controlling the water contamination in future.