基于等温杂交链式反应的沙门氏菌可视化检测技术的研究

发布时间：2018-05-02 22:56

本文选题：肠炎沙门氏菌 + 可视化检测　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：食品贸易作为国际贸易的主要组成部分,在扩大出口创汇、解决粮食供求矛盾的同时,也带来了食源性疾病的发病率和死亡率上升的问题。微生物病原是我国食源性疾病的主要病因,占30%~40%,细菌占微生物病原的81.5%。因此,食源性疾病尤其是由细菌污染引起的食源性疾病是我国食品安全面临的首要问题。我国作为一个食品生产和消费大国,建立快速、准确的食源性病原菌检测技术,对于食品质量监控、保证人民健康至关重要。目前食源性细菌检测的方法主要有三大类:平皿培养法、免疫学检测以及现代分子生物学检测方法。平皿培养法使用的是传统的培养法,不需要复杂的实验仪器并且实验结果可靠,但是检测周期较长,5~7天,而且程序繁琐、费时费力。经典的酶连免疫吸附技术(ELISA)等免疫学检测方法,特异性高、灵敏性高,但是需要多种蛋白质分子(如单克隆抗体)的参与,成本较高,而且对反应环境要求苛刻。新型免疫荧光技术也具有对试剂、仪器选择性高的缺点。分子生物学方法针对的靶标是核酸,但是由于生物体内核酸的含量通常是十分微少的,因此经过温度循环的核酸扩增如聚合酶链式反应(PCR)、实时定量PCR几乎是这类检测方法的前提步骤。但是对于落后地区或者设备资源贫乏的环境,这些检测方法也受到限制。杂交链式反应(HCR)是一种无酶参与、室温条件便可以进行的核酸扩增反应。相对于PCR,HCR并不是对于模板序列的直接扩增,而是间接的对核酸信号进行放大。由于具有无酶参与,反应条件温和等特点,近年来,HCR反应与电化学、荧光信号技术结合已经被广泛应用于核酸、蛋白、病原菌等靶标物质的检测。可视化检测技术,是指检测的结果可以在可见光或者紫外灯下被肉眼观察的实验方法。相对于其他检测方法,因为不需要精密仪器参与,因此检测成本较低,适用场合较广。本实验的目的是以核酸等温扩增反应HCR作为信号放大手段,以肠炎沙门氏菌为检测靶标,建立快速、低成本的检测方法。我们利用NCBI数据库中Blast比对功能筛选出肠炎沙门氏菌核糖体16S rRNA特异性片段作为靶标,设计相应的特异性的抓取探针、检测探针,并以HCR反应作为信号放大手段,分别结合胶体金免疫层析试纸条、微孔板显色这两种方法建立低成本、快速便捷的即时诊断技术(POCT)。在胶体金免疫层析试纸条方法中,我们首先制备了检测试纸条:纳米金颗粒上包被有链霉亲和素、检测线上涂有荧光基团的抗体,质控线上涂有生物素。然后以合成的模拟靶标建立检测模型,并进行相应的条件优化。我们发现HCR反应中最佳启动探针/发夹探针的浓度比例是1:5,修饰探针/不修饰探针的含量比例是9:1,最佳抓取探针浓度是0.3μM并且能够区分靶标沙门氏菌序列与弗氏柠檬酸杆菌、大肠杆菌、阪崎肠杆菌、金黄色葡萄球菌、耶尔森氏菌五种细菌的相似性片段。以合成序列为靶标的检测模型中,未结合HCR反应的检测限是0.31nM,结合HCR反应的方法可以检测到1.76pM,放大倍数为176倍。然后我们将培养的沙门氏菌进行平板计数、并提取其总RNA,结合本方法进行实际样本的检测,计算的LOD值为3×103 CFU mL-1。在获取RNA样本之后,整个检测时间在30min之内,并且结果可以用肉眼判断。在微孔板显色实验中,包被由链霉亲和素的微孔板用于固定具有特异性识别能力的、修饰有生物素分子的抓取探针。靶标序列的存在,使得夹心结构:抓取探针/靶标序列/HCR得以形成,并由于生物素与链霉亲和素之间亲合作用被固定到微孔板上,由于HCR产物长链上标记有荧光基团FITC,可以结合辣根过氧化物酶(HRP)标记的荧光集团抗体anti-FITC-HRP。HRP的存在不仅可以催化显色反应,而且由于自身的高催化活性,达到双重信号放大的能力。实验过程中,我们同样的进行了条件优化:最佳抓取探针的浓度是0.5μM,最佳孵育时是2小时,anti-FITC-HRP的最佳孵育时长是2小时。本实验设计的探针能够区分与靶标有一个碱基差异的序列,以合成序列建立检测模型的检测限是32.6fM。然后我们将培养的沙门氏菌进行平板计数、并提取其总RNA,将提取的细菌RNA用于检测,不仅可以将沙门氏菌与弗氏柠檬酸杆菌、大肠杆菌、金黄色葡萄球菌区分开(非靶标组的信号值均不高于22%),而且针对沙门氏菌的检测限可以达到52.1 CFU mL-1。上述结果表明,本文所建立的杂交链式反应分别结合免疫层析试纸条、微孔板显色用于沙门氏菌可视化检测的方法,具有良好的特异性以及灵敏度。相对于运用电化学、荧光等技术结合HCR反应的检测方法,本文所介绍的两种实验方法在检测的灵敏性方面并不是最佳的,但是由于本实验所运用的胶体金免疫层析试纸条、微孔板显色方法的实验结果都可以用肉眼判断,不需要高端仪器的配合,并且实验过程简单,不需要专业人士的操作,因此比较适用于偏远、资源贫乏的地区用于检测,可以作为POCT诊断试剂进一步的进行开发、应用。
[Abstract]:As the main part of the international trade, the food trade is the main cause of food borne diseases in China, which is the main cause of food borne diseases in China, which accounts for the 30%~40%, and the microbes account for the 81.5%. of the microbial pathogens, especially the food borne diseases. The food borne disease caused by bacterial contamination is the primary problem of food safety in China. As a big food production and consumption country, China is a major food producing and consuming country. It is very important for food quality monitoring and to ensure the health of the people to establish rapid and accurate detection techniques for food borne pathogens. There are three main categories of food borne bacteria detection methods: The method of plate culture, immunology and modern molecular biology test. The plate culture method uses the traditional culture method. It does not need complex experimental instruments and the experimental results are reliable, but the detection cycle is long, 5~7 days, and the program is tedious, time-consuming and laborious. The classic enzyme linked immunosorbent assay (ELISA) and other immunological detection parties It has high specificity and high sensitivity, but requires a variety of protein molecules, such as monoclonal antibodies, with high cost and harsh response to the environment. New immunofluorescence technology also has the disadvantage of high selectivity for reagents and instruments. The target of molecular biology is nucleic acid, but the content of nucleic acid in organism is through. It is often very small, so the real-time quantitative PCR is almost the precondition of this kind of detection through the RNA amplification of temperature cycle like polymerase chain reaction (PCR). But for the backward areas or the poor environment of equipment resources, these detection methods are also restricted. The mixed chain reaction (HCR) is a kind of non enzyme participation, room temperature condition. Compared with PCR, HCR is not a direct amplification of the template sequence, but an indirect amplification of nucleic acid signals. The combination of HCR reaction with electrochemistry and fluorescence signal technology has been widely used in nucleic acid, protein, pathogen and so on in recent years. Detection of target materials. Visual detection technology means that the results of detection can be observed by the naked eye under visible light or ultraviolet light. Compared with other methods, the detection cost is low and the application is wide. The purpose of this experiment is to amplify HCR as a letter by isothermal amplification of nucleic acid. A rapid and low cost detection method was established with Salmonella enteritidis as a target test. We screened the specific fragment of the ribosome 16S rRNA specific fragment of Salmonella enteritidis as a target by using the Blast comparison function in the NCBI database, designed the specific grasping probe, detected the probe, and put the HCR reaction as a signal hand. In a colloidal gold immunochromatographic test paper method, we first prepared the test paper in the colloidal gold immunochromatographic test strip method, which was coated with streptomycin avidin on the gold nanoparticles, and the detection of the antibody on the line with fluorescent groups was detected in the two methods. The control line is coated with biotin. Then the test model is set up with the synthetic target, and the corresponding conditions are optimized. We found that the concentration ratio of the best start probe / hairpin probe in the HCR reaction is 1:5, the content ratio of the modified probe / unmodified probe is 9:1, the best grip concentration is 0.3 mu M and the target salmonk can be distinguished. The sequence is similar to the five bacteria of citric acid bacilli, Escherichia coli, Enterobacter sakazakii, Staphylococcus aureus, and Jerson S bacteria. In the detection model of the synthetic sequence as the target, the detection limit of unbound HCR reaction is 0.31nM, and the method of combining the HCR reaction can detect 1.76pM, and the magnification is 176 times. And then we will The cultured Salmonella was counted, and the total RNA was extracted, and the actual sample was detected with this method. The calculated LOD value was 3 x 103 CFU mL-1. after obtaining the RNA sample, the whole detection time was within 30min, and the result could be judged by the naked eye. The presence of the target sequence makes the sandwich structure: the capture probe / target sequence /HCR is formed, and the affinity between biotin and streptomycin is immobilized to the microporous plate because of the fluorescent group FITC marked on the long chain of the HCR product. The presence of the fluorescent group antibody anti-FITC-HRP.HRP labeled with horseradish peroxidase (HRP) not only catalyzes the color reaction, but also has the ability to achieve double signal amplification due to its high catalytic activity. In the experiment, the same conditions were optimized: the concentration of the best capture probe was 0.5 u M, and the best incubation was 2 hours. The best incubation time of anti-FITC-HRP is 2 hours. The probe designed in this experiment can distinguish the sequence of base difference between the target and the target. The detection limit of the synthetic sequence is 32.6fM., then we will count the Salmonella in the plate, and extract the total RNA, which can be used to detect the bacterial RNA. Salmonella was separated from citric acid bacilli, Escherichia coli and Staphylococcus aureus (the signal value of the non target group was not higher than 22%), and the detection limit for Salmonella could reach 52.1 CFU mL-1.. The results showed that the hybrid chain reaction established in this paper was combined with the immunochromatography test strip and the microplate was used to display color. The method of visual detection of Salmonella has good specificity and sensitivity. Compared with the application of electrochemistry, fluorescence and other techniques combined with HCR reaction, the two methods introduced in this paper are not the best in sensitivity of detection, but the colloidal gold immunochromatography paper strips used in this experiment are micro. The experimental results of the hole plate color display method can be judged by the naked eye. It does not need the cooperation of the high-end instrument, and the experiment process is simple. It does not need the operation of the professionals. Therefore, it is more suitable for remote, poor resource areas for detection. It can be developed and applied as a POCT diagnostic reagent.

【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】：2017
【分类号】：R446.5

【参考文献】