基于单粒子分析方法的水体总菌检测系统关键技术研究

发布时间：2018-01-07 07:25

本文关键词：基于单粒子分析方法的水体总菌检测系统关键技术研究　出处：《中国人民解放军军事医学科学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的:近年来伴随着多种微生物污染问题的爆发,广义的生物安全问题逐渐成为了研究热点。水与空气资源作为微生物污染的主要传播媒介,其广泛的分布以及无可替代的重要程度时刻提醒我们要重视对资源状况的监控与保护。细菌总数作为水中微生物含量的重要指标之一,能够直观的反映水体受污染的程度,因此在许多水质检测标准中均规定将细菌总数作为一项必要的检测项目。在日常生活工作中建立快速准确的水体样本细菌检测方法对生活用水、科研实验、医疗卫生、精密加工质量控制以及水资源的监控与保护,甚至是生物恐怖活动的防治都具有十分重要的意义。目前在该领域应用最多的仍是传统的检测方法,尤其以平板培养法为各国检测的标准方法,新的检测方法的提出为水体细菌的检测提供了许多更快速、更准确的研究思路,但是总体来说这些方法仍处于研究阶段。本文所述课题旨在课题组研究的基础上,进一步掌握单粒子检测技术并将其应用于水体细菌总数的检测。在系统样机研制的过程中积累经验,助力于单粒子检测技术在水体样本检测专项应用方面的推广,为单粒子检测技术的应用提供新的思路。方法和内容:通过对现有的流式检测原理进行剖析,在国外已有的检测技术和相关装备的基础上,提出单粒子水体总菌检测系统关键技术研究和实验装置的研制。主要研究内容及方法包括:I.样本前处理方法研究;通过查阅文献及实验验证,对不同原理的前处理方法进行探索,对比不同染料、不同碳点对细菌的染色效果,确立课题所使用的染色方案在该检测原理下的可行性与准确性;通过前处理方案的确定,为光路系统光源选择、探测通道镜片参数制定提供必要的设计参数。II.光学模块设计与参数优化;将光路模块从总体上分为激发光路与检测光路两部分,通过理论仿真分析,设计激光光路实现对原始光斑的整形从而得到理想的检测光斑;设计检测光路对荧光信号进行选择性接收;在课题中对该系统的光源选择、镜片参数、光路结构三个方面进行系统性优化,并依据前处理方案拟定的染料特性对检测光路参数进行优化处理。III.液流模块设计与参数优化;通过不同设计方案对比,使用不同动力源控制样本液和鞘液,使其在检测区形成由外部鞘液流包裹内部样本液流的同轴流动模式,通过对液流系统效果进行评价分析,优化设计方案与控制参数。课题通过实验探索,确定了具有较高安全性的进样液路及整体流路方案。IV.信号处理模块设计与优化;系统在检测区由激发光斑激发染色后的细菌,经过检测光路对荧光信号进行探测收集,通过光电倍增管将光信号转换为电信号,再经过放大、滤波、AD转换后成为数字信号传递给FPGA主控芯片。课题中针对系统的信号特点进行了相关电路设计及参数优化,绘制PCB板并完成电路系统调试。V.峰值处理算法的提出以及验证;对微米级待测样本粒子经荧光染色后荧光信号强度分布进行建模仿真,探寻待测样本荧光信号的特征,结合系统检测平台的检测需求设计具有针对性的信号处理方法,在Verilog语言环境下实现算法的应用并通过实验验证算法的准确性及稳定性,对算法进行优化。VI.系统结构设计与优化;在系统搭建初期,采用独立的分模块构建方案,将总体系统划分为几个子系统模块,在逐步实现各个模块的拟定功能后,为了提高系统集成度,在控制系统采用以FPGA为核心的控制方案,在硬件电路中围绕FPGA为核心分别构建中心电源板、信号处理板、信号控制板以及微控板为主体的4块直插式电路控制系统,将各独立模块综合集成为一体,优化系统结构。结果:I.光路系统采用设计方案后激光器原始光斑大小约为844*765um,椭圆度约为0.906,经过XY双轴整形透镜组整形后,在检测点位置光斑大小77.8*19.8um,在焦点位置附近光斑变化趋势较小,具有较好的稳定性。II.液流系统分别采用柱塞泵与气压泵作为动力源,解决了样本“死体积”问题引入的污染,避免了“脉动”问题带来的样本回流现象;使样品进样不受样本管体积限制,实现连续检测;大约0.4s即可在鞘液液瓶中建立稳定的气压,电压值约为1.1v,稳定效果较好。III.峰值处理算法重复检测10组样本相对标准偏差为5.36%,并且仅需3个参数即可实现系统信号的快速处理,与现有检测方法相比具有较好的一致性,可满足系统的检测需求。IV.整体系统微球测试表明当进样速度处于0.5ul/s-1ul/s之间时,样本检测结果间差异较小,检测过程更稳定;针对不同浓度样本检测CV值均在2-3.5之间,达到了较好的检测精度。V.系统整体测试人工添加金黄色葡萄球菌10倍梯度浓度结果表明,目前系统最佳检测限为103-106cfu/ml浓度范围,在该检测限内与平皿计数法检测结果具有较好的一致性,在低于该检测限时检测结果明显高于平皿计数法;与现有流式细胞仪的统计结果始终保持较好的一致性。总结与展望:I.在现有样机的基础上,通过对子系统各部分分别优化,进一步提高整体系统的检测灵敏度。II.增加荧光收集通道,在理论上通过该样本前处理方法,可实现对水体细菌总数中死菌与活菌数量分别检测,通过增加检测通道,提高系统单次检测的分析能力。III.优化峰值检测算法,进一步提升算法的灵敏度以及多平台的通用性,提高算法的自适应分析能力。IV.对现有系统结构进行优化,提升系统的集成度与便携性。
[Abstract]:Objective: in recent years, accompanied by a variety of microbes pollution outbreak, bio safety problem of generalized gradually becomes a hot research topic. The main media of water and air resources as microbial contamination, the importance of time its wide distribution and it reminds us to attach importance to the monitoring and protection of resources. As an important index of the total number of bacteria the microbial content in water, is able to reflect the degree of water pollution, so in many water quality testing standards are provisions of the total number of bacteria as a necessary test items. In daily life, water for life, scientific research, medical and health to establish a method for detection of bacteria in water samples quickly and accurately in precision machining quality control and the water resources monitoring and protection, and even has a very important significance for prevention and treatment of bioterrorism. Currently in the lead The detection method is still the most widely used traditional domain, especially with plate culture method as the standard method of national detection, a new detection method is proposed for the detection of water bacteria provides a lot more rapid, more accurate research ideas, but overall these methods are still in the research stage. Based on the topic of this paper aims at research group on the further mastery of single particle detection technology is applied to detect the total number of bacteria in the water. The experience gained in the process of system prototype, and help the single particle detection technology in the application of water samples of the special promotion, to provide new ideas for the application of single particle detection technology. The methods and contents: through the flow detection principle of the existing analysis, based on the existing detection technology and related equipment, the key technologies of detection system of total bacteria in single particle water research and The development of experimental equipment. Including the main research contents and methods: Study on pretreatment method of I. samples; through the literature and experiment, explored the pretreatment method of different principle, comparison of different dyes, the dyeing effect of different carbon on bacteria, establish the accuracy and feasibility of dyeing scheme used in this topic detection principle; determined by pretreatment scheme, selection of light source system, detecting channels provide lens parameters and parameter optimization of design parameters of.II. optical module design will be necessary; optical module includes excitation light path and optical detection part two, through the simulation analysis, the design of laser beam shaping the original spot so as to obtain the ideal spot detection; design of optical detection of fluorescence signal for selectively receiving; selection of the source of the system in the paper, lens parameters, optical path The three aspects of structure system optimization, and based on the characteristics of dye processing scheme to develop the detection optical path to optimize the parameters of.III. flow and parameter optimization design module; through the comparison of different design schemes, using different power control sample solution and the sheath liquid, the coaxial flow mode by external sheath fluid inclusions inside the sample the liquid flow formed in the detection area, were analyzed by flow effect on liquid, optimization design and control parameters. Research by experiments, determined with high safety injection liquid road and road scheme of.IV. signal processing module design and optimization of the overall flow; system in the detection area by the excitation beam excited after dyeing the bacteria, after testing the optical path of the fluorescence signal detected by the photomultiplier tube to collect, convert optical signals into electrical signals, after amplification, filtering, AD converted into digital signal Signal for the FPGA main control chip. The signal characteristics of the system the design and optimization of circuit drawing PCB and complete the circuit board system debugging.V. peak processing algorithm is proposed and verified; on micron sample particles by fluorescent staining after fluorescence signal intensity distribution simulation, to explore the characteristics of sample fluorescence the signal to be measured, combined with the test requirements of design system testing platform is for signal processing methods, algorithms used and the accuracy and stability of the algorithm in the Verilog language environment, optimize the design and optimization of.VI. system structure of the algorithm; in early stage of building the system, the construction scheme of independent modules overall, the system is divided into several sub modules of the system, to gradually realize the function of each module, in order to improve the degree of system integration, in the control system The control scheme based on FPGA as the core in the hardware as the core center were constructed around the FPGA power board, signal processing board, signal control board and control system of the 4 block in-line circuit of micro control board as the main body, each independent module integrated into one, optimize the system structure. Results: the design scheme based on I. the optical system of laser original spot size is about 844*765um, the ellipticity is about 0.906, after the plastic lens group after XY biaxial plastic, in the detection of the position of light spot size 77.8*19.8um, in a small spot change trend of focal position, with using piston pump and pneumatic pump as a power source for better stability of.II. liquid flow system, solution a sample of "dead volume" problems of introducing pollution, avoid the sample ripple problem caused by the reflux phenomenon "; the sample from the sample tube volume limit, realize the continuous detection; about 0.4s can establish a stable pressure in the sheath liquid bottle, the voltage value is about 1.1V, the peak value of.III. stabilization processing algorithm repeated detection of 10 samples the relative standard deviation is 5.36%, and only 3 parameters to realize fast signal processing system, and the existing detection method compared with good consistency, and can meet the the requirements of.IV. system testing system testing shows that when the microsphere injection speed is between 0.5ul/s-1ul/s, the difference between the small sample test results, the detection process is more stable; with different concentration sample CV values were between 2-3.5, achieve the detection accuracy of.V. system better overall test added Staphylococcus aureus 10 times the concentration gradient the current system shows that the best detection limit of 103-106cfu/ml concentration range, the detection results within limits and plate count method have good consistency, low in the The detection limit of detection results were significantly higher than the plate count method; statistics and current flow cytometry results remain consistent. Summary and Outlook: I. based on the existing prototype, respectively through the optimization of each part of the sub system, and further improve the overall system's sensitivity to increase.II. fluorescence collection channel, through processing methods the sample in theory, can respectively realize detection of viable and dead bacteria bacteria in water by increasing the number of detection channels, improve the system of single detection of.III. peak detection algorithm optimization ability, to further improve the algorithm sensitivity and versatility of multi platform, improve the analysis of the existing system structure optimization of.IV. algorithm in adaptive ability, enhance the integration and portability of the system.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R123.1

【参考文献】