基于封闭式卡盒的现场传染病病原体检测系统的研制与应用

发布时间：2018-01-08 06:31

本文关键词：基于封闭式卡盒的现场传染病病原体检测系统的研制与应用　出处：《东南大学》2016年博士论文　论文类型：学位论文

【摘要】：近年来,由于环境变化、自然灾害、物种多样性变化等因素,导致各种病毒引起的传染性疾病不断爆发并重复性出现,如SARS病毒、H1N1、H7N9禽流感病毒、中东呼吸综合征冠状病毒以及这两年来谈之色变的埃博拉病毒,寨卡病毒等。而且随着全球化进程的加快以及传染病病原体传播途径的多变性,使得这些传染病的爆发不再是局部地域出现,更是在全球范围内迅速流行,给人类健康和社会带来极大的危害。因此,对传染性疾病的防控、诊断和治疗已成为全球公共卫生领域的热点和重点。然而传统的传染性病原体检测方法大都只适用于实验室部署环境,具有检测过程分散、繁琐、实验操作复杂、对环境敏感等缺点,并且对实验员的实验技能要求较高。这些弊端都大大限制了传统检测技术在现场传染病检测中的应用。传染病病原体现场快速检测要求具有操作简单、成本低、便携和易于实现自动化等特点,因此,急需寻找一种新的技术平台以满足传染病的现场检测需求。本论文首先利用3D打印技术,设计并制作了封闭式的检测卡盒,在此基础上研发了全自动的便携式核酸检测系统,并对仪器进行了各项性能测试及实际现场传染病的检测,成功地完成了现场传染病从样本输入到结果输出的自动化检测过程。具体内容包括:1.基于3D打印技术的封闭式检测卡盒的设计与制造首先根据病原体核酸检测的实验流程明确了封闭式卡盒的设计需求,提出了基于柔性滑片的封闭式卡盒结构及其内部各功能模块。整个设计与制造过程采用了高精度的3D打印技术及医用级PC-ABS材料,共经历5个批次的快速迭代试制过程,设计和优化了卡盒7个特殊零件的结构,组装完成的卡盒尺寸为140mm× 90 mm × 30 mm。其内部集成移液模块、样本处理及检测区域,样本处理范围为20～1000μL,可同时进行1～6项实时荧光PCR检测。液体操作范围为2～200μL,实测在10 μL和100μL处的移液相对误差分别为1.71%和0.76%,重复性CV值分别为2.3%和0.77%,均优于国标规定。同时,对卡盒内的磁珠法核酸提取及PCR配制过程进行了优化。最后,实验证明了上游核酸提取试剂的挥发对于下游PCR检测并未造成抑制影响,说明了在封闭式卡盒内进行一体化核酸检测是可行的。2.基于封闭式卡盒的现场病原体检测系统的研发以封闭式卡盒为基础,研发了与之配套的自动化驱动系统,其内部由移液控制、磁分离加热、热循环、荧光采集及辅助电路等功能模块组成。其中,移液控制模块的最大操作量程为260 μL,精度为0.118 μL,内含滤芯以防止交叉污染;磁分离加热模块采用倾斜式滑动结构,可程序化控制磁富集的位置,且温控精度为±0.5℃,可在2min内升至目标温度;热循环模块可提供3通道的PCR反应条件,其升降温速度分别为4.6℃/s及4.0℃/s,控制精度为±0.3℃;荧光采集模块提供双色荧光通道(FAM/SYBR Green和Texas Red波长的范围的),采用单色LED作为激发光源,波动性小于1μW,单次荧光信号的采集在2.5 s内完成。仪器整体尺寸为270 mm × 275 mm × 250mm,重量为6.5 kg。现场操作时仅需将带样本的卡盒放入仪器中,并配置实验参数,则可自动完成整个检测流程。3.现场病原体检测系统的性能测试首先使用百日咳病毒质粒DNA进行了核酸提取效率的对比实验,系统和手工提取的效率分别为95.49%和84.33%;使用HBV质粒DNA测试获得了 104 copies/mL的提取限,说明系统的样本处理能力在效率和灵敏度方面均可满足下游PCR检测的需求。同时,分别使用E.coli及HBV病毒的真实临床样本进行了核酸提取对比实验,结果显示采用本系统操作在产量上比手工操作分别高出6.6 ng/μL和0.93 ng/μL。通过平行实验测得本系统的实时荧光PCR检测孔间差异为0.023(CV值),检测限为104copies/mL。最后,使用本系统连续运行20次阴性和阳性样本的间隔实验,测试结果与预期一致,表明系统没有发生批间交叉污染的现象。4.现场传染病检测实验在江苏省疾病防控中心进行了腺病毒的检测实验,并采用罗氏LightCycler 2.0荧光定量PCR仪进行实验对照,获得未知样本的Ct值分别为31.15和31.68,阳参的Ct值分别为27.52和28.01,从扩增曲线形态和Ct值的比较说明两系统的检测结果较为一致。最后,使用本卡盒系统对沙门氏菌及志贺氏菌进行联合检测,并设置卡盒内采用悬滴加样的方式进行PCR体系的配置,同时采用ABI SteponePlus实时荧光定量PCR系统进行实验对照。两系统的扩增曲线形态一致,两路荧光检测(FAMandTexasRed)的Ct值均值差值分别为0.71和0.98左右,仅相差0.29,结果表明卡盒系统在多重检测方面与商用系统的检测结果较为一致。
[Abstract]:In recent years, due to changes in the environment, natural disasters, changes in species diversity and other factors, resulting in various infectious diseases caused by virus outbreak and appear repeatedly, such as SARS H1N1, H7N9 virus, avian influenza virus, coronavirus mers and this year to talk about the Ebola virus discoloration, Zika virus and so on. With the accelerating process of globalization and the variability of the pathogen of infectious diseases spread, the outbreak of the disease is no longer a local region, it is rapidly popular in the global scope, bring great harm to human health and society. Therefore, the prevention and control of infectious diseases, diagnosis and treatment has become the focus in the world in the field of public health. However, most of the traditional infectious pathogen detection method is only applicable to the lab deployment environment, the detection process has dispersed, cumbersome, complex operation experiment, The disadvantage of such sensitive environment, and experimental skills of laboratory technician requirements higher. These disadvantages greatly limits the application of traditional detection techniques in the detection of infectious disease in the field. The pathogens of infectious diseases on-site rapid detection requirements has the advantages of simple operation, low cost, portable and easy to realize automation, therefore, the urgent need to find a new in order to meet the needs of the scene detection technology platform for infectious diseases. This paper first use of 3D printing technology, design and production of the closed box card detection, on the basis of the research and development of portable automatic nucleic acid detection system, and the instruments for the test of the performance test and the practical field of infectious diseases, the successful completion of the site infectious diseases from the sample input to the automatic detection of process output results. The specific contents include: 1. the design and manufacture of box card closed 3D printing technology based on Detection Based on The experimental process of pathogen nucleic acid detection clear design requirements of the enclosed card box, the closed type flexible vane cartridge structure and its internal modules. Based on the design and manufacturing process using 3D printing technology with high accuracy and medical grade PC-ABS material, has experienced rapid iterative development process in 5 batches. The design and optimization of the structure of 7 special parts of the card box, card box size assembled 140mm * 90 mm * 30 mm. the internal integrated pipetting module, sample processing and detection area, sample processing range is 20 ~ 1000 L, and 1 ~ 6 real time fluorescent PCR detection of liquid operation. The range is 2 ~ 200 L, measured at 10 L and 100 L of the pipette relative errors are 1.71% and 0.76%, the repeatability CV values were 2.3% and 0.77%, are better than the national standard. At the same time, the method of nucleic acid extraction and PCR magnetic card box preparation process Optimized. Finally, experiments show that the upstream nucleic acid extraction reagent volatilization on the downstream PCR detection did not cause inhibitory effect, explained in the enclosed card box for the integration of nucleic acid detection is feasible based on the.2. research site blocking pathogen detection system card box with the enclosed card box based automation and development the auxiliary drive system, the internal control by pipetting, magnetic separation heating, thermal cycling, fluorescence collection and auxiliary circuit modules. The maximum operating range shift hydraulic control module is 260 L, the precision is 0.118 L, containing a filter to prevent cross contamination; heating module by inclined magnetic separation sliding structure, capable of controlling the position of magnetic enrichment, and the precision of temperature - 0.5 DEG C, but to the target temperature in 2min; thermal cycle module can provide the PCR reaction conditions of 3 channels, the temperature rise speed Don't is 4.6 DEG /s and 4 DEG /s, the control precision is + 0.3 DEG C; the fluorescence collection module of dual color fluorescence channel (FAM/SYBR Green and Texas Red range of wavelength), using monochromatic LED as excitation source, volatility is less than 1 W, the single fluorescence signal acquisition was completed within 2.5 s. The size of the instrument was 270 mm * 275 mm * 250mm, the weight for the card box 6.5 kg. site operation will only be with the sample into the instrument configuration, and experimental parameters can automatically complete the performance testing of the experimental detection process.3. pathogen detection system of the first use of pertussis virus plasmid DNA was extracted efficiency nucleic acids, efficiency of the system and manual extraction were 95.49% and 84.33%; the use of HBV plasmid DNA was extracted from 104 copies/mL test limit, explains the sample processing ability of the system in terms of efficiency and sensitivity can meet the test needs of downstream PCR Pray. At the same time, the real clinical samples were studied using E.coli and HBV virus were nucleic acid extraction experiments, results show that the yield ratio of manual operation were higher than 6.6 ng/ L and 0.93 ng/ L. through parallel experiments between real-time PCR detection system of the hole is 0.023 by the operation of the system (CV value), the detection limit is 104copies/mL. finally, the continuous operation of the system 20 times of positive and negative sample interval test, the test results were consistent with the expected.4. field indicates the phenomenon of infectious disease detection system without cross contamination between batch experiments of adenovirus detection in Jiangsu Province Center for Disease Control and prevention, and the use of Roche LightCycler 2 fluorescence quantitative PCR instrument experiment obtained the unknown sample Ct values were 31.15 and 31.68, Yang joined the Ct values were 27.52 and 28.01, compared with that from the amplification curve shape and Ct value Two more consistent detection system results. Finally, the use of the card box system for joint detection of Salmonella and Shigella, and set the PCR card system using the hanging drop and the way in the box configuration, at the same time the control experiment by using real-time quantitative PCR system ABI. SteponePlus amplification curve form two the two fluorescence detection (FAMandTexasRed) of the Ct mean value of difference is about 0.71 and 0.98, a difference of only 0.29. The results show that more consistent detection card box system and commercial system in the multiple detection results.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TP274;R446

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