基于上转发光免疫层析霍乱弧菌多重检测技术研究

发布时间：2018-04-01 02:11

本文选题：霍乱弧菌　切入点：上转发光免疫层析　出处：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：1.研究背景及目的霍乱是由霍乱弧菌引起的一种急性腹泻性传染病,其发病急,传染性强,死亡率高,是《中华人民共和国传染病防治法》规定的甲类传染病之一。现在霍乱疫情仍然时有报道,严重威胁着人民群众的健康,快速、简便、准确的检测霍乱尤为重要。现已证实霍乱弧菌共有超过200种血清群,其中O1群与O139群为主要致病群,其他型别几乎不会造成疾病流行。因此准确检测O1与O139群具有很大的现实意义。现有的霍乱检测手段或多或少都存在一些问题,比如有的需要实验操作人员丰富的经验,有的需要依靠精密的仪器设备,有的需要严密的实验设计,有的容易造成结果误判,有的只能定性判断,有的实验耗时较长,有的敏感性不是很高,有的实验成本较高,这些不足都抑制了霍乱弧菌临床快速检验的发展。2.研究方法免疫层析作为经典快速检验技术,操作简便,耗时很短,仅使用制备完成的拭纸,直接将样品加至样品垫,静置一段时间后,即可在视窗肉眼观察结果。然而经典的免疫层析技术过于依赖操作者的个人经验,且只能定性判断结果,这些缺点都制约了免疫层析的发展。将上转换发光材料与免疫层析相结合,可以很大程度上避免这一问题。上转发光免疫层析检测样品时,没有杂光干扰,背景干净,既保留了经典免疫层析技术的优点,如操作简便,设备轻巧,实验耗时很少,又解决了经典免疫层析的问题,比如结果判定依赖肉眼观察,易造成假阳性或假阴性,不能定量判断等,是一种具有临床快速检验前景的新技术。本论文通过研究,利用基于上转发光免疫层析技术(Up-converting phosphor technology-based immunochromatographic assay,UPT-ICG),采用双抗体夹心的方法分别制备了3种拭纸——检测小川型的9B12单抗自配拭纸;检测霍乱O1的9F11单抗自配拭纸;检测霍乱主要流行株的M-(9F11+mAb2)+P-(9F11+mAb1);还制备完成了可以检测不同分辨度霍乱弧菌的三靶标拭纸。三靶标拭纸可以同时检测霍乱弧菌O1群小川型、霍乱弧菌O1群与霍乱弧菌O139群,三条检测带之间互不干扰,基本可以达到对霍乱弧菌主要致病群的甄别。通过对拭纸样品垫成分优化、结合垫抗体量与标记条件优化、分析膜抗体量优化,现在这3种单靶标拭纸与1种三靶标拭纸可以达到很好的敏感性、线性、精密性与特异性。经过现场评价,4种拭纸均可以达到预期实验结果,检测准确率很高。3.研究结果我们制备了霍乱弧菌O1群小川型单克隆抗体9B12、霍乱弧菌O1群稻叶型单克隆抗体9F11;并且利用这两种单克隆抗体,结合已有的霍乱弧菌O139群抗体mAb1与mAb2制备了三种基于上转发光免疫层析的单靶标拭纸,可以分别检测霍乱弧菌O1群小川型、霍乱弧菌O1群与霍乱弧菌流行株——即O1群与O139群。其中检测霍乱弧菌O1群小川型的拭纸M-9B12+P-9B12、检测霍乱弧菌O1群的拭纸M-9F11+P-9F11的敏感性均为102cfu/ml,检测霍乱弧菌流行株的拭纸M-(9F11+mAb2)+P-(9F11+mAb1)敏感性为103cfu/ml。检测其他食源性菌,如沙门氏菌、副溶血弧菌、致病型大肠埃希菌、李斯特菌等时,均为阴性反应,可见这三种单靶标拭纸具有良好的特异性。在中国疾病预防控制中心进行的临床分离株现场评价实验,共检测菌株78株,包括霍乱弧菌O1群小川型15株,稻叶型12株,彦岛型12株(与小川型有血清凝集的共5株),O139群15株,非O1非O139群24株。针对霍乱弧菌O1群小川型的M-9B12+P-9B12检测小川型覆盖度为100%,检测各型霍乱弧菌的准确率为98.7%。针对霍乱弧菌O1群的拭纸M-9F11+P-9F11检测霍乱O1群的覆盖度为100%,检测各型霍乱的准确率为100%。在三种单靶标拭纸的基础上,我们研发成功了一种可以同时检测不同分辨度霍乱弧菌的三靶标拭纸,在定性判断检测霍乱弧菌样品的血清群、定量检测其浓度的基础上,可以进行初步分型。根据出现峰值的T带,判定待检样品是霍乱弧菌O1群小川型,或是霍乱弧菌O1群,或是霍乱弧菌O139群。这在国内外尚属首创。该三靶标拭纸检测霍乱弧菌的敏感性可达103cfu/ml,与其他食源性菌,如沙门氏菌、副溶血弧菌、致病型大肠埃希菌、李斯特菌等无交叉反应,特异性好,在中国疾病预防控制中心进行临床分离株现场评价实验,其覆盖度为100%,检测各型霍乱的准确率为100%。我们研发的4种基于稀土纳米上转发光免疫层析技术的高灵敏霍乱弧菌检测拭纸具有操作简便,仪器易携带,实验耗时短,可以定性定量判定实验结果,敏感性与特异性理想等优点,是很有潜力的临床快速检验方法。4.讨论在现有的霍乱弧菌检测方法不能完全满足临床快速检验的要求,存在各种各样缺陷的情况下,基于稀土纳米上转发光免疫层析技术的拭纸研发,很大程度上可以弥补这一问题。使用上转发光免疫层析拭纸检测菌株时,仅需要使用已配制好的样品处理液(本论文使用的是碱性蛋白胨水)稀释样品,然后使用加样枪吸取样品加至拭纸加样孔内,静置15min后使用UPT生物传感器检测,即可获得实验结果。一次实验操作涉及的拭纸、样品处理液为事前制备完成的;生物传感器体积轻巧,便于携带;实验操作非常简单,对实验操作者没有任何技术要求,易于推广,非常适合临床快速检验及在基层卫生医疗中使用,对霍乱疫情的快速发现、及时处理、得当防控都具有非常重要的意义。
[Abstract]:1. background and objective cholera is caused by Vibrio cholera is an acute infectious disease of diarrhea, the incidence of acute, highly infectious, high mortality, is one of the "infectious diseases prevention and treatment of infectious diseases in People's Republic of China. Now the cholera epidemic > provisions are reported, a serious threat to people's health, fast and convenient accurate detection of cholera, Vibrio cholerae is particularly important. It has been confirmed that there are more than 200 kinds of serum group, O1 group and O139 group as the main pathogenic group, other types can hardly cause epidemic disease. Therefore, accurate detection of O1 and O139 group has great practical significance. Cholera existing detection methods have some problems, such as some experimental operating personnel with rich experience, some need to rely on sophisticated equipment, some require rigorous experimental design, easy to cause the results of some false positives, some can only set The judgment of some experimental time-consuming, some sensitivity is not very high, some experimental research methods.2. development cost is high, these problems are inhibited the rapid detection of Vibrio cholerae clinical immune chromatography as classic rapid detection technology, simple operation, time is very short, only completed with a wipe, directly added to the sample the sample pad, after standing for a long time, can be observed in the naked eye window the results. However immunochromatography classic is too dependent on the experience of the operator, and can only be qualitative judgment, these shortcomings have restricted the development of immunochromatographic. The upconversion luminescence materials and immunochromatography combined, can largely avoid this a problem. To luminescence immunochromatography test samples, no stray light interference, clean background, not only retains the advantages of classical immunochromatographic technique, such as simple operation, light equipment, very time-consuming experiment Less, but also solve the problems such as classical immune chromatography, determination of the naked eyes, easily lead to false positive or false negative, not quantitative judgment, is a new technology with a rapid test clinical prospect. Through the research, the use of transgenic technology based on layer analysis on chemiluminescence immunoassay (Up-converting phosphor technology-based immunochromatographic assay. UPT-ICG), using the method of double antibody sandwich were prepared 3 kinds of wiping paper - Detection Ogawa 9B12 monoclonal antibody with swabs; detection of cholera O1 9F11 monoclonal antibody with swabs; detection of cholera strains M- (9F11+mAb2) +P- (9F11+mAb1); also completed the preparation can detect different resolution the degree of Vibrio cholerae three target wipes. Three target wipes can simultaneously detect Vibrio cholerae O1 serotype Ogawa, Vibrio cholerae O1 and Vibrio cholerae O139 detection, three with no mutual interference between the basic. In order to achieve the main pathogenic Vibrio cholerae group screening. Based on the wiping paper sample pad composition optimization, combination pad antibody quantity and labeling conditions optimization, analysis of membrane antibody quantity optimization, now the 3 single target swabs and 1 kinds of three target wipes can achieve very good sensitivity, linearity, precision and specificity after on-site evaluation, 4 kinds of wiping paper can reach the expected results, the detection accuracy rate is very high.3. our results for monoclonal antibody 9B12 against Vibrio cholerae O1 serotype Ogawa by monoclonal antibody 9F11 against Vibrio cholerae O1 serotype Inaba; and the use of these two kinds of monoclonal antibodies, three based on the turn light immunochromatography single target swabs were prepared with the Vibrio cholerae O139 antibody mAb1 and mAb2 were detected respectively, Vibrio cholerae O1 serotype Ogawa, Vibrio cholerae O1 and Vibrio cholerae strains of O1 group and O139 group. The detection of Cholera Cholerae O1 serotype Ogawa wipe M-9B12+P-9B12, the sensitivity of detection of Vibrio cholerae O1 group M-9F11+P-9F11 swabs were 102cfu/ml for detection of Vibrio cholerae strains were M- (9F11+mAb2) +P- paper (9F11+mAb1) sensitivity for 103cfu/ml. detection of other foodborne pathogens, such as Salmonella, Vibrio parahaemolyticus, pathogenic Escherichia coli, Lester bacteria, were negative, showing that these three single target swabs had good specificity. In clinical isolates of site evaluation experiment China Center for Disease Control and prevention, were detected in 78 strains, including choleraic vibrio O1 serotype 15 strains, 12 strains of rice leaf type, hikojima 12 strains (serum agglutination and Ogawa total 5 strains), 15 strains of group O139, non O1 and non O139 group. 24 strains of M-9B12+P-9B12 detection for Vibrio cholerae Ogawa serotype Ogawa O1 coverage is 100%, the detection rate of each type of Vibrio cholerae 98.7%. in Huo Chaos wipes M-9F11+P-9F11 group O1 detection of Vibrio cholera O1 group coverage is 100%, the detection rate of each type of cholera 100%. based on three kinds of single target wipes on, we successfully developed a can simultaneously detect different resolution of Vibrio cholerae three target swabs, determine the detection of Vibrio cholerae samples in the qualitative and quantitative detection of serum group, based on its concentration, can make a preliminary classification. According to the peak value of T, to determine the sample to be detected is Vibrio cholerae O1 serotype Ogawa, and Vibrio cholerae O1, or Vibrio cholerae O139. This is the first at home and abroad. The three target samples paper detection of Vibrio Cholera sensitivity up to 103cfu/ml, and other food borne bacteria, such as Salmonella, Vibrio parahaemolyticus, pathogenic Escherichia coli and other bacteria, no cross reaction to Lester, good specificity, in Chinese disease prevention and control center for clinical isolates of the scene The experimental evaluation and its coverage is 100%, the detection rate of various types of cholera 100%. we developed 4 kinds of luminescence immunochromatography technology based on rare earth nano on high sensitive detection of Vibrio cholerae wipes instrument has the advantages of simple operation, easy to carry, the short time, can determine the qualitative and quantitative experimental results, the sensitivity and specificity of ideal the advantages of.4. clinical rapid test methods have the potential to discuss completely meet the clinical requirements in rapid detection of Vibrio cholerae not existing detection methods, there are various kinds of defects in the case of R & d wipe paper converting phosphor immunochromatography nano based on great extent can make up for this problem. Chemiluminescence immunoassay the use of paper chromatography swab was detected, only need to use liquid sample has been prepared (this paper is using alkaline peptone water) dilution of the sample, and then use the sample suction gun The sample is added to the wiping paper sample adding hole, static 15min after using UPT biosensor, obtained experimental results. An experimental operation involving a wipe, liquid sample preparation prior to complete system; biosensor is lightweight, easy to carry; the operation is very simple, no technical requirements for the operator is easy to popularize, very suitable for clinical use and rapid test in the primary health care, to quickly find the cholera epidemic, timely treatment, has a very important significance for prevention and control properly.

【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】：R516.5;R446.6

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