细菌直接PCR和双引物策略鉴定16S rRNA基因技术的建立

Bacterial direct PCR of 16S rRNA gene identification using dual primer strategy

目的探索一种基于16S rRNA基因的快速鉴定细菌方法,为临床诊断治疗及耐药菌的分子遗传分析提供科学依据。方法对卫生部室间质评菌株和临床病人标本分离培养纯菌落,用双蒸水稀释菌落,然后直接以菌液为模板优化反应体系PCR扩增16S rRNA基因片段,再测序扩增片段。将测序结果在细菌Ribosomal数据库中进行同源性比对,根据序列同源性鉴定病原细菌。结果本实验鉴定的卫生部质评菌株结果与卫生部报告结果一致。本实验能够一次性鉴定出临床病人标本分离的菌株。结论本研究优化了一种免纯化细菌核酸直接PCR鉴定细菌16S rRNA基因的方法。本研究建立的基于病原细菌16S rRNA基因鉴定方法可用于细菌的快速诊断。

Objective This study explored an identified method based on 16S rRNA gene sequence analysis of occupational disease with bacterial infection to provide a scientific basis for clinical diagnosis and treatment of drug-resistant and molecular genetic analysis. Methods The strains from Ministry of Health external quality assessment and isolated from clinical patients were diluted with distilled water, and then the diluted samples were directly detected by optimizing PCR amplification of 16S rRNA gene fragment. The amplified fragments were performed DNA sequencing. The sequences were blasted in bacteria Ribosomal sequence database for the identification of the pathogenic bacteria according to the sequence homology. Results The results for strains from the Ministry of Health quality assessment identified by new method were concordant with the report of Ministry of Health. The strain that were isolated from clinical specimens of patients can be identified directly by this method. Conclusion This study optimized a PCR and 16S rRNA gene identification method without purification of bacterial nucleic acid. The pathogen identification method based on the 16S rRNA gene developed in this study may be used for rapid diagnosis of bacteria.