本文選題：細(xì)菌 + 診斷�。� 參考：《河北醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:通過檢測部分臨床常見肺部感染的細(xì)菌菌株(腸球菌、鏈球菌、金黃色葡萄球菌ATCC29312、ATCC29213、ATCC25923)在實(shí)驗(yàn)條件下生長產(chǎn)生的細(xì)菌揮發(fā)性物質(zhì)(Bacterial volatile compounds BVCs),通過光聲光譜痕量分析儀(photoacoustic spectroscopy trace analyzer PASTA1001)檢測BVCs,篩選出各細(xì)菌產(chǎn)生的能區(qū)別于其他細(xì)菌菌株的特殊BVCs峰,探索呼出氣在肺部感染性疾病的診斷中的臨床應(yīng)用提供實(shí)驗(yàn)數(shù)據(jù)。方法:氣體光聲光譜技術(shù)是利用光聲效應(yīng)測量氣體濃度的一種間接吸收光譜技術(shù),其基本原理是當(dāng)光聲池內(nèi)的待測氣體分子吸收特定波長的光輻射后被激發(fā)到高能態(tài),在氣體分子從高能態(tài)向低能態(tài)的無輻射躍遷過程中,引起了氣體的溫度和壓強(qiáng)變化,如果對入射光進(jìn)行強(qiáng)度調(diào)制或波長調(diào)制,光聲池內(nèi)氣體便會(huì)呈現(xiàn)出與調(diào)制頻率相同的溫度變化,進(jìn)而導(dǎo)致壓強(qiáng)的變化,當(dāng)調(diào)制頻率在聲頻范圍內(nèi)時(shí),便產(chǎn)生聲音信號(hào),即光聲信號(hào)。不同物質(zhì)的光聲光譜圖是不一樣的,基于這一原理,我們利用基于硅MEMS(Microelectromechanical Systems)懸臂梁增強(qiáng)型可調(diào)諧激光光聲光譜技術(shù),通過PASTA1001光聲光譜痕量分析儀分別在5小時(shí)、24小時(shí)、48小時(shí)檢測體外固定培養(yǎng)基(增菌培養(yǎng)基)培養(yǎng)腸球菌、鏈球菌、金黃色葡萄球菌ATCC29312、ATCC29213、ATCC25923的密閉培養(yǎng)罐頂空氣體中的揮發(fā)性代謝產(chǎn)物,通過設(shè)立空白對照組,實(shí)驗(yàn)組,并重復(fù)實(shí)驗(yàn)三次,采用Origin8軟件繪制光聲光譜圖,從中篩選出各種病原菌的特征性的BVCs峰。利用線性回歸及相關(guān)分析同一細(xì)菌在不同時(shí)間段及不同細(xì)菌在同一時(shí)間是否存在差異。結(jié)果:通過比較空白培養(yǎng)基對照與加入致病菌的培養(yǎng)基可發(fā)現(xiàn):增菌培養(yǎng)基無細(xì)菌接種5小時(shí)、24小時(shí)、48小時(shí)檢測光聲光譜圖直線回歸R值0.999,輪廓圖沒有差異,說明基線穩(wěn)定,不影響實(shí)驗(yàn)結(jié)果,且重復(fù)實(shí)驗(yàn)共3次后,結(jié)果相同,具有良好的儀器檢測穩(wěn)定性。金黃色葡萄球菌不同菌株ATCC29312、ATCC29213、ATCC25923在24小時(shí)、48小時(shí)測的揮發(fā)性代謝指紋圖譜歸一化處理后,重復(fù)培養(yǎng)檢測3次,直線相關(guān)系數(shù)r值分別是0.999、0.994、0.997,能再現(xiàn)檢測圖譜,說明重復(fù)培養(yǎng)相同細(xì)菌,實(shí)驗(yàn)可重復(fù)性強(qiáng)。金黃色葡萄球菌不同菌株ATCC29312、ATCC29213、ATCC25923和臨床鏈球菌、腸球菌24小時(shí)檢測的幅值較5小時(shí)變化直線回歸趨勢R分別是0.229、0.376、0.446、0.485、0.323,說明產(chǎn)生揮發(fā)性物質(zhì)種類比較多,通過圖譜比對差異也明顯。金黃色葡萄球菌不同菌株ATCC29312、ATCC29213、ATCC25923和臨床鏈球菌、腸球菌48小時(shí)檢測的幅值較24小時(shí)變化直線回歸趨勢R分別是0.960、0.904、0.994、0.990、0.979,說明產(chǎn)生揮發(fā)性物質(zhì)種類比較少,趨于穩(wěn)定狀態(tài),通過圖譜比對發(fā)現(xiàn)差異不明顯。金黃色葡萄球菌不同菌株ATCC29312、ATCC29213、ATCC25923在5小時(shí)、24小時(shí)、48小時(shí)檢測的光聲光譜圖通過比較,發(fā)現(xiàn)在5小時(shí)檢測的1030、1040、1060 cm-1出現(xiàn)有差異的峰值,在24小時(shí)檢測的1030、1048、1080 cm-1出現(xiàn)有差異的峰值,在48小時(shí)檢測的1210、1246 cm-1出現(xiàn)有差異的峰值,1060cm-1出現(xiàn)ATCC25923的特征峰,1246cm-1出現(xiàn)ATCC29312的特征峰,且重復(fù)實(shí)驗(yàn)三次后,結(jié)果相同,具有良好的穩(wěn)定性。金黃色葡萄球菌ATCC25923、臨床鏈球菌、腸球菌在5小時(shí)、24小時(shí)、48小時(shí)檢測的光聲光譜圖通過比較,發(fā)現(xiàn)在5小時(shí)檢測的1048和1080cm-1出現(xiàn)有差異的峰值,在24小時(shí)檢測,發(fā)現(xiàn)在1033-1048、1055-1090 cm-1出現(xiàn)有差異的峰值,1075 cm-1出現(xiàn)腸球菌的特征性雙峰,48小時(shí)未發(fā)現(xiàn)新的差異波段,且重復(fù)實(shí)驗(yàn)三次后,結(jié)果相同,具有良好的穩(wěn)定性。細(xì)菌濃度越高,產(chǎn)生的揮發(fā)性代謝產(chǎn)物圖譜峰值越高。結(jié)論:部分體外培養(yǎng)的細(xì)菌通過代謝產(chǎn)生的BVCs,通過光聲光譜技術(shù)檢測發(fā)現(xiàn)金黃色葡萄球菌ATCC25923,腸球菌有特征的BVCs峰,通過其有差異的BVCs峰能區(qū)別實(shí)驗(yàn)室培養(yǎng)的腸球菌,鏈球菌和金黃色葡萄球菌ATCC29312、ATCC29213、ATCC25923。光聲光譜技術(shù)通過BVCs指紋圖譜可能用于實(shí)驗(yàn)室條件下培養(yǎng)細(xì)菌的快速鑒定。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R446.5

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