可视化LAMP法等温快速检测病原菌与恒温纸质微流体芯片构建的研究

发布时间：2017-12-26 21:00

本文关键词：可视化LAMP法等温快速检测病原菌与恒温纸质微流体芯片构建的研究　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:病原菌的快速检测和准确鉴定在临床实验室检测,生物战,食品工业和环境监测中是非常重要的。传统的细菌鉴定微生物学方法包括选择性培养,生物化学和血清特性检测等。这些传统的方法几乎完全依赖于使用特定的琼脂培养基分离和挑选活的病原细菌。在实验室中最常用的鉴定方法是培养,进而基于微生物的表型和生长培养基进行细菌的鉴定。然而,这些用于细菌分析的培养实验需要几天或几周才能提供准确的结果。事实上,在过去的几十年里,许多快速的方法被开发来减少检测时间。到目前为止,快速的检测方法包括酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA),侧流免疫测定法和聚合酶链反应(polymerase chain reaction,PCR)等,其检测时间可以减少到10 24 h和4 6 h,灵敏度可以达到102-106 cfu/ml。然而,这些测试需要成本较高的仪器和熟练的专业操作人员,进而限制了其广泛应用,特别是在发展中国家和地区。在现场检测(Point-of-Care Test,POCT)及基层普及使用中它们不是最优的选择。因此发展使用一种快速、简单和高特异性的检测新技术对于病原微生物的检测诊断具有重要的临床意义。核酸的恒温扩增技术主要特点是核酸扩增从开始到结束都是在一个恒定的温度下进行。与普通PCR相比,它降低了温控设备的成本,只需要一个普通的恒温装置,同时也减少了反应的步骤和时间。因此在POCT诊断中,核酸恒温扩增比普通PCR更适合。在当前核酸的等温扩增方法中,环介导等温扩增(loop-mediated isothermal amplification,LAMP)法是一种结果较稳定的方法,利用一种Bst DNA聚合酶和特异引物在温度不变(65℃左右)和较短时间(30-60min)的条件下即可对目的片段进行扩增,并且拥有较高的灵敏性和特异性。此外,它只需要一个恒温装置,从而使检测的费用明显减少。所以,本课题使用LAMP技术构建一种简单、快速的病原菌核酸检测法。早在1990年,Manz A和Widmer团队首次指出在一块以平方厘米为单位的小芯片上进行样品制备、生化反应、结果检测分析的微流体芯片技术。其拥有高效率、低损耗(包括检测用时、标本量、试剂量),分析微型化和高通量化等特点,并且通过结合传感或样品前处理模块,为以后的现场检测带来了方便。纸质材料,有着来源丰富、价格低廉、可循环、不易变性、易处理、易运送、易化学修饰、以及与生物具有较好的相容性等特点,和现代印刷技术相结合可以制备出集分离、分析和检测一体的微流体装置。然而目前市场上的纸质微流体芯片主要用来检测免疫蛋白而在核酸检测上使用较少。其可能的原因是由于核酸普通PCR扩增需要循环的变化温度,因而在纸芯片上操作存在一定难度,限制了它的使用。本课题旨在探索在纸质微流体芯片上进行LAMP等温扩增并对扩增产物进行检测,提高纸质微流体芯片在现场诊断中的使用价值。目的:本项目拟充分整合LAMP技术和纸质微流体芯片技术的特色与优势,设计和构建LAMP-纸质微流体芯片,基于LAMP法构建一种快速、简单的病原菌检测方法,便于现场或野外的早期诊断和早期治疗。方法:1.病原微生物及其耐药基因的可视化LAMP快速检测的构建。1)以铜绿假单胞菌为首的病原微生物收集,并对亚胺培南耐药性进行药敏实验。2)采用简易水煮法快速提取DNA。3)针对铜绿假单胞菌的Opr L和Opr D2基因设计LAMP引物。4)可视化LAMP反应体系的构建。5)可视化LAMP检测的特异性和灵敏性分析。6)临床标本的检测。7)统计学分析Opr D2阴性和阳性菌株与亚胺培南的耐药性之间的差异。2.用于等温扩增核酸的纸质微流体芯片的设计和制作。1)在生物活性纸片上对病原微生物进行LAMP扩增检测。2)用于等温扩增核酸的纸质微流体芯片的设计。3)选用whatman#1滤纸进行纸质微流体芯片手工简易制作。4)使用场发射扫描电镜对制作的纸质微流体芯片的微观结构观察。3.基于链霉亲和素-生物素化LAMP体系的构建和条件的优化。1)基于链霉亲和素-生物素化LAMP反应体系的构建:根据生物素和链霉亲和素的共价偶联作用,将生物素化的LAMP产物固定在由纳米金标记链霉亲和素的纸芯片表面。2)利用荧光定量PCR仪对其反应体系中最佳温度,时间,d NTPs浓度,Bst DNA大片段聚合酶浓度,Mg SO4浓度,Betaine浓度以及内外引物浓度进行选择和优化。3)利用荧光定量PCR仪对基于链霉亲和素-生物素化LAMP反应的灵敏性和特异性进行分析。4)临床分离株的检测。结果:1.成功的简化了提取病原微生物DNA的操作,缩短了实验时间。并且成功构建了一个快速、灵敏、简便的检测耐亚胺培南铜绿假单胞菌的Opr L和Opr D2基因的可视化LAMP法。其特异性好,灵敏度(17.41μg/L)比传统PCR高10倍。以传统方法(细菌培养,VITEK 2系统,琼脂稀释法)为金标准,对临床标本Opr L和Opr D2基因同时进行可视化LAMP法和常规PCR法检测。对Opr L基因,可视化LAMP法检测的灵敏性和特异性都为100%,普通PCR法分别为94.9%和87%。对Opr D2基因,可视化LAMP法检测的灵敏性和特异性分别为75%和737%,普通PCR法分别为70%和73.7%。统计学分析表明,Opr D2阳性和Opr D2阴性菌株对亚胺培南耐药菌株具有统计学差异。(P0.05)。2.以铜绿假单胞菌为例,成功的在纸片上对其核酸DNA进行了LAMP扩增。3.成功的对核酸等温扩增的纸质微流体的设计和简易的制作。获得国家的实用新型专利(用于等温扩增核酸的纸质微流体,ZL 201420583698.X),国家的发明专利(一种利用纸质微流体进行核酸等温扩增的方法,201410528335.0)。4.成功的构建了基于链霉亲和素-生物素化LAMP反应体系,为下一步在微流体芯片上固定和信号放大检测垫定了扎实的实验基础。并对其LAMP反应体系条件进行了优化:得到最佳反应扩增的温度为65℃,反应扩增时间为40min,反应液中Mg SO4最佳浓度为6 m M,Bst DNA聚合酶浓度为0.32U/μl、d NTPs混合液的工作浓度为1.2m M,b-FIP/BIP内引物浓度最佳浓度为1.6μM,F3/B3外引物最佳浓度为0.4μM,Betaine的最佳浓度为0.6m M。以铜绿假单胞菌为例,基于链霉亲和素-生物化LAMP法使用荧光定量PCR仪检测分析,其特异性好,灵敏度(0.402μg/L)比可视化LAMP法高100倍,比普通PCR高1000倍。使用该方法检测铜绿假单胞菌的临床分离株,其检出率与可视化LAMP法相当(14/14,100%),而常规PCR法(13/14,93%)。结论:1.可视化LAMP法用于快速检测病原微生物及其耐药基因具有检测时间短、过程简单、灵敏度和特异性高、反应结果肉眼可辨等特点。本课题以铜绿假单胞菌Opr L和Opr D2基因为例,成功建立了可视化LAMP检测技术,使检测结果的识别更容易。统计学分析显示在亚胺培南耐药方面,Opr D2阴性菌株具有更高的风险。早期识别Opr L和Opr D2基因将有助于我们更好地对IRPA感染选择抗生素治疗方案,这将降低成本和时间。这种诊断方法更适合用于现场和基层医疗机构的使用。2.微流体芯片技术是指在一块以平方厘米为单位的小芯片上进行样品制备、生化反应、结果检测分析的技术。其拥有高效率、低损耗(包括检测用时、标本量、试剂量),分析微型化和实验高通量化等特点,并且通过结合传感或样品前处理模块,为以后的现场检测带来了方便。纸作为生物传感材料有许多优势,它的使用使生物传感器更加小巧,方便易于携带,检测成本低,是未来生物传感发展的趋势。在本研究中将LAMP技术与纸质微流体芯片技术进行整合,成功设计和制作了基于核酸等温扩增的纸质微流体芯片,便于下一步在野外或现场对感染性病原微生物的早期定性或定量检测。
[Abstract]:Background: rapid detection and accurate identification of pathogenic bacteria are very important in clinical laboratory testing, biological warfare, food industry and environmental monitoring. The traditional microbiological methods for bacterial identification include selective culture, biochemistry and detection of serum characteristics. These traditional methods are almost entirely dependent on the isolation and selection of living pathogenic bacteria using a specific agar medium. The most commonly used method in the laboratory is to identify the bacteria based on the microbial phenotype and growth medium. However, the cultivation experiments used for bacterial analysis will take days or weeks to provide accurate results. In fact, in the past few decades, many fast methods have been developed to reduce detection time. So far, a rapid detection method of enzyme linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA), lateral flow immunoassay and polymerase chain reaction (polymerase chain reaction, PCR), the detection time can be reduced to 1024 h and 46 h, the sensitivity can reach 102-106 cfu/ml. However, these tests require high cost instruments and skilled professional operators, which, in turn, limit their wide range of applications, especially in developing countries and regions. They are not the best choice in Point-of-Care Test (POCT) and at the grassroots level. Therefore, it is of great significance to develop a rapid, simple and highly specific detection technology for the detection and diagnosis of pathogenic microbes. The main characteristic of nucleic acid constant temperature amplification is that the nucleic acid amplification is carried out at a constant temperature from the beginning to the end. Compared with the ordinary PCR, it reduces the cost of the temperature control equipment, requires only an ordinary thermostat device, and reduces the reaction steps and time. Therefore, in the POCT diagnosis, the nucleic acid constant temperature amplification is more suitable than the common PCR. In the isothermal nucleic acid amplification method, loop mediated isothermal amplification (loop-mediated isothermal, amplification, LAMP) method is a more stable outcome, at constant temperature by using a Bst DNA polymerase and specific primers (65 DEG C) and short time (30-60min) under the condition of objective can be amplified, and have high sensitivity and specificity. In addition, it only needs a thermostat device, so that the cost of detection is significantly reduced. Therefore, this subject uses LAMP technology to construct a simple and rapid detection method for nucleic acid of pathogenic bacteria. As early as 1990, Manz A and Widmer team first pointed out that microfluidic chip technology is applied to sample preparation, biochemical reaction and result detection and Analysis on a small chip with square centimeters. It has the characteristics of high efficiency and low loss (including detection time, quantity of samples, reagent quantity), analysis of miniaturization and high-throughput quantification, etc., and combined with sensor or sample pre-processing module, it brings convenience for future field detection. The paper material, has a rich source, low price, can be recycled, not easydenatured, easy processing, easy to transport, easy chemical modification, and biological compatibility is good, and the combination of modern printing technology can be prepared by microfluidic device in isolation, analysis and detection of development. However, the current market paper microfluidic chips are mainly used to detect immune proteins and are less used in nucleic acid detection. The possible reason is that the common PCR amplification of nucleic acids requires a cyclic change in temperature, so it is difficult to operate on the paper chip, which limits its use. The purpose of this study is to explore the LAMP isothermal amplification on paper microfluidic chips and detect the amplified products, so as to improve the application value of paper microfluidic chips in field diagnosis. Objective: this project aims to fully integrate the characteristics and advantages of LAMP technology and paper-based microfluidic chip technology, the design and construction of LAMP- paper microfluidic chip, establish a rapid and simple method for pathogen detection based on LAMP method, to facilitate on-site or field early diagnosis and early treatment. Methods: 1. the construction of visual LAMP for the rapid detection of pathogenic microbes and their resistance genes. 1) the pathogenic microorganism, headed by Pseudomonas aeruginosa, was collected and the drug sensitivity test was carried out on imipenem resistance. 2) quick extraction of DNA by simple boiling method. 3) the LAMP primers were designed for the Opr L and Opr D2 genes of Pseudomonas aeruginosa. 4) the construction of visual LAMP reaction system. 5) the specificity and sensitivity analysis of visual LAMP detection. 6) detection of clinical specimens. 7) statistical analysis of the differences between Opr D2 negative and positive strains and imipenem resistance. 2. the design and production of a paper microchip for isothermal amplification of nucleic acid. 1) LAMP amplification of pathogenic microbes on bioactive paper. 2) the design of a paper microfluidic chip for isothermal amplification of nucleic acids. 3) the whatman#1 filter paper is used to make the paper microfluidic chip handmade and easy to make. 4) the microstructure of a paper microchip produced by field emission scanning electron microscopy (SEM) was observed. 3. the construction and optimization of the LAMP system based on streptomycin biotinylated. 1) based on the construction of streptavidin biotinylated LAMP reaction system, based on the covalent coupling of biotin and streptavidin, the biotinylated LAMP product was immobilized on the surface of the gold chip labeled with streptavidin. 2) fluorescent quantitative PCR was used to select and optimize the best temperature, time, D NTPs concentration, Bst DNA large fragment polymerase concentration, Mg SO4 concentration, Betaine concentration and the concentration of internal and external primers in the reaction system. 3) the sensitivity and specificity of the streptomycin - biotinylated LAMP reaction were analyzed by the fluorescence quantitative PCR instrument. 4) detection of clinical isolates. Results: 1. successfully simplified the operation of extracting pathogenic microorganism DNA and shortened the experiment time. And successful construction
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R446.5

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