基于单细胞拉曼技术的口腔病原微生物快速鉴别研究

Rapid Identification of Oral Pathogens Microorganism Based on Single-cell Raman

目的:评价单细胞拉曼技术对3种口腔病原微生物的快速分类效能。方法:(1)培养变异链球菌UA159、白色念珠菌ATCC10231和粪肠球菌ATCC29212至稳定期,绘制生长曲线评估生长状态;(2)测量对数和稳定期菌株单细胞拉曼图谱,使用主成分和随机森林分析方法分析光谱。结果:(1)3个菌株的对数期和稳定期均可被拉曼技术快速区分。准确率分别为:99.6%、99.86%、99.60%;(2)单细胞拉曼技术能够快速精确区分稳定期的3种实验菌株,准确率高达99.68%;(3)通过随机森林算法分析得到3种稳定期实验菌株之间的拉曼差异峰分别为:1126~1128 cm^(-1)、736~744 cm^(-1)、1330~1440 cm^(-1)、778~785 cm^(-1)、1001~1003 cm^(-1)和1431~1481 cm^(-1)。其生物学标志分别为:蛋白质、胸腺嘧啶、脂质、胞嘧啶、尿嘧啶、苯丙氨酸、蛋白质。结论:单细胞拉曼光谱可高效区分物种的生长时期和菌株类别。

Objective:To evaluate the efficiency of single-cell Raman technique for rapid classification of three kinds of oral pathogens microorganism.Methods:Streptococcus mutans UA159,Candida albicans ATCC10231,and Enterococcus faecalis ATCC29212 were cultured in each medium respectively until the stationary phase.Cells were sampled and treated as below:(1)inoculated culture was monitored by the optical density at 600 nm to measure bacterial growth.(2)inoculated culture was sampled at logarithmic and stationary phase respectively to measure the Raman spectra.Results:Firstly,the logarithmic phase and stationary phase of three strains could be rapidly distinguished by Raman technique.And the classification accuracy rates of random forest were 99.6%,99.86%,and 99.60%.Secondly,Raman based method could discriminate different oral microbes in stationary phase,and the specificity in classification was 99.68%.Lastly,Raman biomarkers for the classification were 1126-1128 cm^(-1)(protein),736-744 cm^(-1)(thymine),1330-1440 cm^(-1)(lipid),778-785 cm^(-1)(cytosine,uracil),1001-1003 cm^(-1)(phenylalanine),and 1431-1481 cm^(-1)(Marker protein 1451),respectively.Conclusion:Single-cell Raman could be used to differentiate between growth phases of a single species and strain types of difference species.