快速定量检测细菌的FCM系统研制及性能评估  

Development and performance evaluation of flow cytometry for rapid quantitative detection of bacteria

作　　者：严心涛[1,2] 王策 裴智果[1] 刘珏[3] 武晓东 YAN Xin-tao;WANG Ce;PEI Zhi-guo;LIU Jue;WU Xiao-dong(CAS Key Lab of Bio-Medical Diagnostics,Suzhou Institute of Biomedical Engineering and Technology,Chinese Academy of Sciences,Suzhou 215163,China;Academy for Engineering & Technology,Fudan University,Shanghai 200433,China;Suzhou New District Water Supply Development Co. Ltd.,Suzhou 215011,China)

机构地区：[1]中国科学院苏州生物医学工程技术研究所中科院生物医学检验技术重点实验室,江苏苏州215163 [2]复旦大学工程与应用技术研究院,上海200433 [3]苏州高新区自来水有限公司,江苏苏州215011

出　　处：《光学精密工程》2019年第6期1245-1254,共10页Optics and Precision Engineering

基　　金：国家863高技术研究发展计划资助项目(No.2011AA02A106);苏州市科技局民生科技计划资助项目(No.SS201726)

摘　　要：为了实现对饮用水中细菌快速定量检测,建立了基于流式细胞术的高通量定量检测系统。对该系统的信号采集系统、绝对计数方法以及在细菌检测方面的综合性能进行了研究和评估。根据饮用水中典型细菌的荧光染料及其荧光激发光谱特点,介绍了流式细胞术快速检测细菌的工作原理及硬件平台。通过简化细菌的荧光信号强度计算模型,评估了信号采集系统的信噪比。建立了基于流量传感器的绝对计数方法,将检测系统与以参比微球法进行绝对计数的BDLSRFortessa进行了一系列对比实验和统计学分析,测试和评估了检测系统在饮用水中细菌检测的综合性能水平。实验结果表明:对于4MHz宽带的荧光信号,信号采集系统的信噪比可达86dB;对于一定浓度内的微球,系统对它测试cv值低于2%,与BD仪器测试结果的相关系数高达0.9996,对等比例稀释的微球测试线性度高达0.9998,最低可检测细菌浓度可达102particles/mL;系统对E.coli和S.aureus含量测试结果的cv值均低于7%,与BD仪器测试结果的相关系数均高于0.9959,两仪器测试结果的相对误差均在4%以内。该仪器能实现对细菌的高精度快速定量检测,为饮用水中典型细菌快速检测仪器的开发提供了参考。To realize rapid quantitative detection of bacteria found in drinking water,a high-throughput quantitative detection system based on flow cytometry was established. The system s signal acquisition system,absolute counting method,and comprehensive performance for bacterial detection were investigated and evaluated. Firstly,according to the fluorescent dyes of typical bacteria found in drinking water and their fluorescence excitation spectra,the working principle and hardware platform of the flow cytometer for rapid bacterial detection were established. Secondly,the signal-to-noise ratio of the signal acquisition system was evaluated by simplifying the calculation model for signal strength of bacterial fluorescence. Next,a calculation method based on the flow sensor method was established for absolute counting. Finally,a series of statistically analyzed control experiments were performed with the self-developed instrument alongside BD LSR Fortessa with absolute reference according to the reference microsphere method. The performance of the self-developed instrument for the detection of bacteria in drinking water was comprehensively tested and evaluated. The experimental results indicated the followings: for a 4 MHz wideband fluorescent signal,the signal-to-noise ratio of the signal acquisition system can reach 86 dB;for microspheres in a certain concentration range,the cv value of the system test results is less than 2%,the correlation coefficient with the test results of the BD instrument is as high as 0.999 6,the linearity of the equivalent dilution microsphere test is up to 0.999 8,and the lowest detectable bacterial concentration is10 2 particles/mL;for E.coli and S.aureus bacteria,the cv value of the test results of the system is less than 7%,the correlation coefficient with the test results of the BD instrument exceeds 0.995 9,and the relative deviation is within 4%. The instrument is capable of rapid quantitative detection of bacteria with a high-precision,providing a reference for the development of rapid d

关 键 词：流式细胞术 细菌 饮用水 微生物 自来水 绝对计数 

分 类 号：TH79[机械工程—仪器科学与技术] Q93-33[机械工程—精密仪器及机械]