基于16S rDNA快速鉴定细菌的PCR测序方法的建立及其进化关系分析  

Analysis on the establishment of PCR sequencing method for rapid identification of bacteria based on 16S rDNA and its evolutionary relationship

作　　者：宋路萍 杨振苹 王斌 邢体坤 郝一楠 江魁 王亚萍 黄帅 张静静 SONG Luping;YANG Zhenping;WANG Bin;XING Tikun;HAO Yi’nan;JIANG Kui;WANG Yaping;HUANG Shuai;ZHANG Jingjing(Research and Development Center,Hualan Biological Eengineering,Inc.,Xinxiang 453003,China;Quality Control Department,Hualan Genetic Engineering Co.,Ltd.,Xinxiang 453000,China)

机构地区：[1]华兰生物工程股份有限公司研发中心,河南新乡453003 [2]华兰基因工程有限公司质量控制部,河南新乡453000

出　　处：《中国医药科学》2020年第17期86-91,共6页China Medicine And Pharmacy

基　　金：“重大新药创制”科技重大专项(2018ZX09736010)。

摘　　要：目的建立基于16S rDNA快速鉴定细菌的PCR测序方法。方法通过一组16S rDNA通用引物扩增得到基因组全长,PCR产物经纯化后直接测序分析。利用BLAST软件从GenBank数据库中搜索相关菌株的16S rDNA全序列,采用Clustal X软件进行多序列比对和同源性分析,确定细菌的种属。结果实验利用引物建立了16S rDNA全长序列分析方法,未知菌种和已知菌株通过PCR方法获得约1500bp的全长序列。比对分析已知菌株测序结果与预期标准序列完全一致,证实建立的PCR方法结果可靠。利用建立的PCR法,对实验室从污染物中分离得到不同未知菌株进行鉴定,成功确定了这些菌株的种属。结论建立的基于16S rDNA的PCR方法是可行的,可快速准确地检测及鉴定细菌种类,尽早发现细菌污染,对污染制品输注后的针对性治疗方面具有潜在的应用价值。Objective To establish a PCR sequencing method for rapid identification of bacteria based on 16S rDNA.Methods The full length of the genome was obtained by amplification with a set of 16S rDNA universal primers.PCR products were purified and analyzed by direct sequencing.BLAST software was used to search the 16S rDNA full sequence of related strains from GenBank database.Clustal X software was used to carry out multi-sequence alignment and homology analysis to determine the species and genus of bacteria.Results In the experiment,a 16S rDNA full-length sequence analysis method was established by using primers.A full-length sequence of about 1500 bp by PCR was obtained by unknown strains and known strains.The sequencing results of the known strains by comparative analysis were completely consistent with the expected standard sequences,in which the established PCR method was confirmed.The established PCR method was used to identify different unknown strains isolated from pollutants in the laboratory,and the species and genera of these strains were successfully determined.Conclusion The established PCR method based on 16S rDNA is feasible,which quickly and accurately detects and identifies bacterial species,finds bacterial contamination as soon as possible,and has potential application value in targeted treatment after infusion of contaminated products.

关 键 词：细菌污染 16S rDNA 多聚酶链式反应 芽孢杆菌 

分 类 号：R446.5[医药卫生—诊断学]