16S rDNA序列分析在临床不常见细菌鉴定中的初步应用

目的建立细菌16S rDNA序列分析技术并探讨其在临床不常见细菌鉴定中的应用价值。方法收集经全自动微生物分析系统鉴定其可靠率在99%及以上的常见细菌10株和常规方法难以鉴定或少见菌17株,通过PCR扩增16S rDNA v3-v4区基因片段并进行序列测定,序列与16SpathDB 2.0数据库上已知细菌的16S rDNA序列进行比对分析确定细菌种属。当v3-v4序列片段不能明确鉴定细菌时,结合16S rDNA长片段测序及管家基因dnaJ和rpoB的序列做进一步分析。结果经16S rDNA v3-v4区序列分析,10株(100%)临床常见菌都能鉴定到种水平,其可靠性大于98.0%。17株不常见菌株中,10株(58.8%)细菌只与1种菌相似性大于98.0%,成功鉴定到单个种水平;6株(35.3%)细菌与不止1种菌的相似比大于98.0%;1株菌与已知菌相似性在96.0%~98.0%之间只能鉴定到属(棒状杆菌)。进一步结合16S rDNA长片段及管家基因dnaJ和rpoB的序列分析,所有菌株均可鉴定到单个种水平。结论结合16S rDNA和管家基因序列分析,可用于临床常见及不常见细菌的分子鉴定。

Identification of infrequent medical bacteria by 16S rDNA sequence

Objective To establish bacterial 16S rDNA sequencing technique and apply in the identification of infrequent medical bacteria. Methods Total of 10 common clinical isolates identified by automated microbiology system with a probability 〉199% and 17 unidentifiable or infrequent bacteria were collected. V3-V4 regions of 16S rDNA were amplified, sequenced and submitted to 16SpathDB for bacteria identification. Strains with a low level of discrimination were further analyzed with 16S rDNA long fragment and other housekeeping genes （dnaJ and rpoB） sequences. Results Based on sequence analysis of V3-V4 regions of 16S rDNA, All common strains could be successfully identified to species with similarity〉98.0%. Among the 17 infrequent strains, 10 isolates （58.8%） were determined as a single bacterial species having 〉98.0% nucleotide identity, 6 isolates （35.3%） were determined as more than one bacterial species having 〉98.0% nucleotide identity, 1 isolate was determined to the genus level of Corynebacterium. Combined with 16S rDNA long fragment, dnaJ and rpoB sequences analysis, all strains could be identified to single species level. Conclusion The approach based on sequence analysis of 16S rDNA and housekeeping genes could be successfully applied for common and infrequent medical bacteria identification.