流式细胞术检测结核分枝杆菌死活菌混合样本中活菌比例的研究  

Study on the detecting of living bacteria proportion in living and dead Mycobacterium tuberculosis mixed samples by flow cytometry

作　　者：竺澎波[1] 王伟亮 谢贝[1] 李华 孟繁荣[1] 王楠[1] 杨瑜[1] 刘志辉[1] 张言斌[1] Zhu Pengbo;Wang Weiliang;Xie Bei;Li Hua;Meng Fanrong;Wang Nan;Yang Yu;Liu Zhihui;ZhangYanbin(Guangzhou Chest Hospital,Guangzhou 510095,China;Tuberculosis Department,The Fourth People's Hospital of Foshan,Foshan 528000,China)

机构地区：[1]广州市胸科医院,510095 [2]佛山市第四人民医院结核科,528000

出　　处：《国际医药卫生导报》2020年第11期1502-1507,共6页International Medicine and Health Guidance News

基　　金：广东省自然科学基金(2014A030313755);“艾滋病和病毒性肝炎等重大传染病防治”国家科技重大专项(2018ZX10715004);广州市高水平临床重点专科和培育专科建设项目(穗卫函[2019]155号);广东省转化医学创新平台建设项目(省卫函[2018]1254号)。

摘　　要：目的探讨利用基于荧光素二乙酸酯(FDA)的流式细胞术检测结核分枝杆菌(MTB)混合菌液中活菌比例的性能,为应用流式细胞术定量检测与分析MTB异质性耐药方法的创建提供实验依据。方法将10株1麦氏浊度MTB纯培养活菌悬液和其相应的1麦氏浊度85℃加热30 min处死的MTB菌液按一定比例混合得到MTB死活菌混合菌液(其中活菌比例分别为100%、75%、50%、25%和0%),应用FDA分别对其进行染色后,流式细胞仪检测其平均荧光强度(MFI),求其与对应100%活菌样本MFI值的比值来模拟MTB异质性耐药检验中的FDA敏感指数,双变量直线回归分析得到敏感指数与实际活菌比例之间的直线回归方程,并利用该方程计算另外10组死活菌混合菌液样本中MTB活菌比例。结果(1)死菌与活菌MTB样本经FDA染色后MFI值比较差异有统计学意义(P<0.05),该方法用于区分死菌与活菌的界值为1834,用该界值鉴定其余20个死、活未知的MTB样本,其检测的灵敏度为100%、特异度为100%。FDA染色能够明确鉴别MTB的死、活。(2)10组已知活菌比例的MTB死活菌混合菌液样本经FDA染色后各比例之间MFI值比较差异有统计学意义(P<0.05),敏感指数(Y)与混合样本中活菌比例(X)之间的直线回归方程:Ŷ=0.986X+0.016,该直线回归方程差异有统计学意义(P=0.000)。(3)应用这种染色方法对其余50个MTB死活菌混合菌液样本进行检测,计算活菌比例与实际活菌比例之间差异无统计学意义(P>0.05),基于FDA染色方法的流式细胞术可用于MTB死活菌混合菌液样本中活菌比例的检测。结论通过基于FDA染色的流式细胞术具有准确检测MTB混合菌液样本中活菌比例的潜力,该方法在MTB异质性耐药检测中将具有广阔的应用前景。Objective To explore the performance of flow cytometer(FCM)based on fluorescein diacetate(FDA)in detecting the proportion of living bacteria in the mixed samples of Mycobacterium tuberculosis(MTB),and to provide experimental basis for the establishment of quantitative detection and analysis of MTB heterogeneity resistance by FCM.Methods The mixture of dead and living MTB(the proportion of living MTB was 100%,75%,50%,25%,and 0%,respectively)were obtained by mixing the 10 strains of MTB pure culture and its corresponding dead MTB heated at 85℃for 30 min.The samples were dyed by FDA and the average fluorescence intensity(MFI)was detected by flow cytometer.The MFI ratio between MTB mixture samples and its corresponding 100%living MTB samples were calculated.The linear regression equation between the MFI ratio and proportion was obtained by bivariate linear regression analysis.Then the equation was used to calculate the proportion of living MTB in the other 10 groups of dead and living MTB mixture samples.Results(1)There was statistically significant difference in FDA MFI between living and dead bacteria(P<0.05).The cutoff value of this method which was used to distinguish between dead and living bacteria was 1834.The cutoff value was used to distinguish the other 20 clinical samples of which the living or dead status were unknown.The sensitivity of the method for detecting living bacteria was 100%and the specificity was 100%.(2)The MFI value difference among 5 different living bacteria proportion of 10 groups of MTB mixture was statistically significant(P<0.05).The MFI ratio was calculated and the linear regression equation between the MFI ratio(Y)and the living bacteria proportion of MTB mixture(X)was established:Ŷ=0.986X+0.016.The linear regression equation was statistically significant(P=0.000).(3)The difference between the calculated living bacteria proportion and the actual living bacteria proportion was not statistically significant.So the flow cytometry based on FDA staining can be used to detect the propor

关 键 词：流式细胞术 结核分枝杆菌死活菌混合菌液 荧光素二乙酸 

分 类 号：R52[医药卫生—内科学]