细菌挥发性代谢产物的研究

发布时间：2018-04-26 18:43

本文选题：细菌 + 诊断　；参考：《河北医科大学》2016年硕士论文

【摘要】：目的:通过检测部分临床常见肺部感染的细菌菌株(肠球菌、链球菌、金黄色葡萄球菌ATCC29312、ATCC29213、ATCC25923)在实验条件下生长产生的细菌挥发性物质(Bacterial volatile compounds BVCs),通过光声光谱痕量分析仪(photoacoustic spectroscopy trace analyzer PASTA1001)检测BVCs,筛选出各细菌产生的能区别于其他细菌菌株的特殊BVCs峰,探索呼出气在肺部感染性疾病的诊断中的临床应用提供实验数据。方法:气体光声光谱技术是利用光声效应测量气体浓度的一种间接吸收光谱技术,其基本原理是当光声池内的待测气体分子吸收特定波长的光辐射后被激发到高能态,在气体分子从高能态向低能态的无辐射跃迁过程中,引起了气体的温度和压强变化,如果对入射光进行强度调制或波长调制,光声池内气体便会呈现出与调制频率相同的温度变化,进而导致压强的变化,当调制频率在声频范围内时,便产生声音信号,即光声信号。不同物质的光声光谱图是不一样的,基于这一原理,我们利用基于硅MEMS(Microelectromechanical Systems)悬臂梁增强型可调谐激光光声光谱技术,通过PASTA1001光声光谱痕量分析仪分别在5小时、24小时、48小时检测体外固定培养基(增菌培养基)培养肠球菌、链球菌、金黄色葡萄球菌ATCC29312、ATCC29213、ATCC25923的密闭培养罐顶空气体中的挥发性代谢产物,通过设立空白对照组,实验组,并重复实验三次,采用Origin8软件绘制光声光谱图,从中筛选出各种病原菌的特征性的BVCs峰。利用线性回归及相关分析同一细菌在不同时间段及不同细菌在同一时间是否存在差异。结果:通过比较空白培养基对照与加入致病菌的培养基可发现:增菌培养基无细菌接种5小时、24小时、48小时检测光声光谱图直线回归R值0.999,轮廓图没有差异,说明基线稳定,不影响实验结果,且重复实验共3次后,结果相同,具有良好的仪器检测稳定性。金黄色葡萄球菌不同菌株ATCC29312、ATCC29213、ATCC25923在24小时、48小时测的挥发性代谢指纹图谱归一化处理后,重复培养检测3次,直线相关系数r值分别是0.999、0.994、0.997,能再现检测图谱,说明重复培养相同细菌,实验可重复性强。金黄色葡萄球菌不同菌株ATCC29312、ATCC29213、ATCC25923和临床链球菌、肠球菌24小时检测的幅值较5小时变化直线回归趋势R分别是0.229、0.376、0.446、0.485、0.323,说明产生挥发性物质种类比较多,通过图谱比对差异也明显。金黄色葡萄球菌不同菌株ATCC29312、ATCC29213、ATCC25923和临床链球菌、肠球菌48小时检测的幅值较24小时变化直线回归趋势R分别是0.960、0.904、0.994、0.990、0.979,说明产生挥发性物质种类比较少,趋于稳定状态,通过图谱比对发现差异不明显。金黄色葡萄球菌不同菌株ATCC29312、ATCC29213、ATCC25923在5小时、24小时、48小时检测的光声光谱图通过比较,发现在5小时检测的1030、1040、1060 cm-1出现有差异的峰值,在24小时检测的1030、1048、1080 cm-1出现有差异的峰值,在48小时检测的1210、1246 cm-1出现有差异的峰值,1060cm-1出现ATCC25923的特征峰,1246cm-1出现ATCC29312的特征峰,且重复实验三次后,结果相同,具有良好的稳定性。金黄色葡萄球菌ATCC25923、临床链球菌、肠球菌在5小时、24小时、48小时检测的光声光谱图通过比较,发现在5小时检测的1048和1080cm-1出现有差异的峰值,在24小时检测,发现在1033-1048、1055-1090 cm-1出现有差异的峰值,1075 cm-1出现肠球菌的特征性双峰,48小时未发现新的差异波段,且重复实验三次后,结果相同,具有良好的稳定性。细菌浓度越高,产生的挥发性代谢产物图谱峰值越高。结论:部分体外培养的细菌通过代谢产生的BVCs,通过光声光谱技术检测发现金黄色葡萄球菌ATCC25923,肠球菌有特征的BVCs峰,通过其有差异的BVCs峰能区别实验室培养的肠球菌,链球菌和金黄色葡萄球菌ATCC29312、ATCC29213、ATCC25923。光声光谱技术通过BVCs指纹图谱可能用于实验室条件下培养细菌的快速鉴定。
[Abstract]:Objective: to detect the volatile substances (Bacterial volatile compounds BVCs) produced by bacterial strains (Enterococcus, Streptococcus, Staphylococcus aureus ATCC29312, ATCC29213, ATCC25923) in some clinical common pulmonary infections (Bacterial volatile compounds BVCs), and photoacoustic spectroscopy trace analyz (photoacoustic spectroscopy trace analyz) Er PASTA1001) detection of BVCs, screening out the special BVCs peak that different bacteria can distinguish from other bacterial strains, and explore the clinical application of exhalation in the diagnosis of pulmonary infectious diseases. Method: gas photoacoustic spectroscopy is an indirect absorption spectrum technique using photoacoustic effect to measure gas concentration. Its basic original When the gas molecules in the photoacoustic pool absorb the specific wavelength of the light radiation, they are excited to the high energy state. In the process of the non radiation transition of the gas molecules from the high energy state to the low energy state, the temperature and pressure change of the gas are caused. If the intensity modulation or wavelength modulation is carried out on the incident light, the gas in the photoacoustic pool will be presented and modulated. The change in the temperature of the same frequency leads to the change of the pressure. When the modulation frequency is in the sound frequency range, the sound signal is produced, that is, the photoacoustic signal. The photoacoustic spectra of different substances are different. Based on this principle, we use the tunable laser photoacoustic spectrum based on the cantilever Liang Zengqiang type of the silicon MEMS (Microelectromechanical). Technology, the volatile metabolites of Enterococcus, Streptococcus, Staphylococcus aureus, Staphylococcus aureus ATCC29312, ATCC29213, ATCC25923 in closed culture tank headspace gas were detected by PASTA1001 photoacoustic spectrum analyzer at 5 hours, 24 hours and 48 hours respectively, and the experimental group was set up by setting up a blank control group. And repeat the experiment three times, using Origin8 software to draw the photoacoustic spectrogram to screen out the characteristic BVCs peak of various pathogenic bacteria. By linear regression and correlation analysis, whether there is difference between the same bacteria at different time and different bacteria at the same time. It was found that there was no bacterial inoculation for 5 hours, 24 hours, 48 hours, 48 hours and 0.999, and no difference in contour map, which showed that the baseline was stable and did not affect the experimental results. After repeated experiments, the results were the same, the results were the same, and the stability of the instrument was good. The different strains of Staphylococcus aureus, ATCC29312, ATCC29213 After the normalization of the volatile metabolic fingerprint of 24 hours and 48 hours, the ATCC25923 was repeated for 3 times. The R value of the linear correlation coefficient was 0.999,0.994,0.997, which could reproduce the detection atlas, indicating the repetition of the same bacteria, and the reproducibility of the experiment was strong. The different strains of Staphylococcus aureus, ATCC29213, ATCC25923 and pro. The amplitude of 24 hours detection of Streptococcus bed and Enterococcus was 0.229,0.376,0.446,0.485,0.323, which showed that R was 0.229,0.376,0.446,0.485,0.323, indicating that there were more kinds of volatile substances, and the different strains of Staphylococcus aureus, ATCC29312, ATCC29213, ATCC25923 and clinical Streptococcus, 48 hours of Enterococcus, were detected. The linear regression trend of the measured amplitude compared with 24 hours was 0.960,0.904,0.994,0.990,0.979, which showed that the variety of volatile substances was less and tended to be stable, and the difference was not obvious by the spectrum comparison. The photoacoustic light and light detected by different strains of Staphylococcus aureus, ATCC29312, ATCC29213, ATCC25923, were detected at 5 hours, 24 hours, and 48 hours. By comparison, it was found that there was a difference peak in the 103010401060 cm-1 detection in 5 hours, and there was a difference peak in the 103010481080 cm-1 detection in 24 hours. There was a difference peak in the 12101246 cm-1 of the 48 hour detection, the 1060cm-1 appeared the characteristic peak of ATCC25923, and the 1246cm-1 appeared the characteristic peak of ATCC29312, and repeated the experiment. After three times, the results were the same, with good stability. The photoacoustic spectra of Staphylococcus aureus ATCC25923, clinical Streptococcus, Enterococcus in 5 hours, 24 hours and 48 hours were compared, and there was a difference peak in 1048 and 1080cm-1 in 5 hours, and the difference between 1033-10481055-1090 cm-1 and 24 hours was found to be poor. The difference peak value, 1075 cm-1 of Enterococcus characteristic Shuangfeng, 48 hours did not find the new difference band, and after repeated experiments three times, the results are the same, with good stability. The higher the concentration of bacteria, the higher the peak of the peak of the spectrum of volatile metabolites. The spectrum technique detected Staphylococcus aureus ATCC25923 and Enterococcus has the characteristic BVCs peak, which can distinguish the laboratory culture of Enterococcus through its different BVCs peaks, Streptococcus and Staphylococcus aureus ATCC29312, ATCC29213, ATCC25923. photoacoustic spectroscopy may be used in laboratory conditions to develop bacteria fast through the BVCs fingerprint. Speed identification.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R446.5

【参考文献】