快速检测单增李斯特菌、登革病毒和西尼罗病毒的超顺磁免疫层析技术研究

发布时间：2018-01-09 23:18

本文关键词：快速检测单增李斯特菌、登革病毒和西尼罗病毒的超顺磁免疫层析技术研究　出处：《暨南大学》2015年博士论文　论文类型：学位论文

【摘要】：单增李斯特菌可引起人、畜的李氏特菌症,感染后表现为免疫力低下、免疫缺陷、脑膜炎、败血症、孕妇流产或早产、内脏器官感染脓肿及坏死。该菌以食物为传播媒介,致死率高达30%~50%,是最致命的食源性致病菌之一。人类感染登革病毒和西尼罗病毒后在临床上可引起以发热、皮肤和黏膜出现淤点或淤斑、不同脏器的损害和出血以及低血压、休克等为特征的综合征,这两种病毒均以蚊为传播媒介,是常见的虫媒病毒,感染虫媒病毒对人类健康造成严重威胁。随着快餐食品、生鲜食品消费人群的不断增加,单增李斯特菌感染引起的食品安全事件在美国、欧洲等地区不断出现;随着以登革病毒、西尼罗病毒等为代表的虫媒病毒疫源地的扩大,这两种病毒的感染已引起世界各国的广泛关注。上述三种病原微生物引起的人和动物疾病已经成为全球性公共卫生问题,加强对这些病原体的快速检测具有重要的现实意义。针对上述三种病原体建立早期、快速和准确的检测方法不仅对于及时控制患者病情、提高患者生存机率起着重要作用,同时对于口岸卫生检疫机构及时监测污染源、甄别和隔离病人、严防污染食品或疫情传入国内至关重要。近年来随着纳米技术、免疫标记技术和侧向层析技术的发展,一种综合了纳米超顺磁技术、免疫层析技术以及磁力定量测定技术的新型免疫检测方法--超顺磁免疫层析技术问世,以其高灵敏、定量化、重复性强、线性范围宽等特点日益受到关注。本研究旨在建立超顺磁免疫层析技术,研制单增李斯特菌超顺磁免疫层析快速检测卡以及同时检测登革病毒和西尼罗病毒的超顺磁免疫层析联合检测卡。本研究首先研制了单增李斯特菌的超顺磁免疫层析快速检测卡。通过对12种单增李斯特菌抗体喷膜及超顺磁微球标记配对,筛选出1对能用于检测单增李斯特菌的抗体对;通过建立标准曲线,确定了单增李斯特菌超顺磁免疫层析卡的检测灵敏度约为104CFU/m L,线性范围为104~108CFU/m L;特异性检测发现该检测卡特异性较好,虽然与部分非致病李斯特菌属的绵阳李斯特菌(L.ivanovii)和西尔李斯特菌(L.seeligeri)存在交叉反应,但仍具有很高的特异性;在73份不同来源的食品样品检测中,超顺磁免疫层析法和荧光PCR法的检测结果100%匹配。第二部分研制了可同时检测登革病毒和西尼罗病毒的超顺磁免疫层析联合检测卡。通过对8种登革病毒抗体、4种西尼罗病毒抗体喷膜和超顺磁微球标记配对,分别筛选出1对能用于检测登革病毒和用于检测西尼罗病毒的抗体对;通过建立标准曲线,确定了检测登革病毒的灵敏度为10ng/m L,线性范围为10ng/m L~10000ng/m L,检测西尼罗病毒的灵敏度为100ng/m L,线性范围为100ng/m L~10000ng/m L;特异性检测发现联合检测卡检测登革病毒和西尼罗病毒时相互之间具有部分交叉反应,检测黄热病毒和乙型脑炎病毒时没有交叉反应;通过对100份口岸送检实际样品检测(其中25份登革病毒核酸阳性样品,75份阴性样品),发现联合检测卡检测登革病毒的准确率为80.0%,假阳性率29.3%,检测西尼罗病毒的假阳性率为89.9%。本研究成功建立了针对单增李斯特菌、登革病毒和西尼罗病毒快速检测的超顺磁免疫层析法,该方法的灵敏度和线性范围均优于常规免疫层析法;检测单增李斯特菌特异性与荧光PCR法相当;操作简便,快速,是一种可靠的新型检测方法。本研究成功筛选出的单增李斯特菌和登革病毒检测抗体对,均可作为诊断试剂开发所用配对抗体的技术储备,针对单增李斯特菌和登革病毒检测的超顺磁免疫层析技术部分已获得发明专利和实用新型专利保护,有利于该技术方法进一步向疾病预防部门、口岸检验检疫机构、医院等单位推广应用,提高这些病原体现场初筛的技术能力。
[Abstract]:Listeria bacteria can cause Lester, Lee special bacterium disease animal, after infection showed immunity, immunodeficiency, meningitis, septicemia, miscarriage or premature birth, viscera abscess and necrosis. The infection of bacteria in food for the media, death rate as high as 30%~50%, is one of the most deadly foodborne pathogens of human. Dengue virus infection and Siniro virus in clinic can cause fever, skin and mucosa petechia or ecchymosis, different organ damage and hemorrhage, hypotension, shock and other characteristics of the syndrome, the two viruses are mosquito borne arbovirus infection, is a common, serious threat to human arbovirus health. With the increasing of fast food, fresh food consumer groups, Lester Listeria bacteria infection caused by food safety incidents continue to occur in the United States, Europe and other regions; with dengue virus, Expand the Siniro virus as the representative of arbovirus foci, these two kinds of virus infection has caused widespread concern in the world. By the above three kinds of pathogens of human and animal diseases has become a global public health problem, has important practical significance to strengthen the rapid detection of these pathogens. Set up early for the above three kinds of pathogens rapid and accurate detection method, not only for the timely control of the disease, which plays an important role in improving the probability of survival, at the same time for the port health and quarantine agencies timely monitoring of pollution sources, screening and isolation of patients, to prevent contamination of food or epidemic spread to the home is very important. In recent years with the development of nanotechnology, immune markers and lateral tomography, a combination of superparamagnetic nano technology, immunochromatography quantitative determination technology and magnetic immune detection model Methods: the advent of superparamagnetic magnetic immunochromatographic technology, with its high sensitive, quantitative, reproducible, wide linear range has been paid more and more attention. The purpose of this study is to establish the super paramagnetic immunochromatography technology development of Listeria bacteria Lester superparamagnetic immunochromatographic detection card and simultaneous detection of dengue virus and Siniro virus the combined detection of superparamagnetic magnetic immunochromatography card. This study first developed Listeria bacteria Lester superparamagnetic immunochromatographic test card. The 12 Listeria bacteria Lester antibody spraying film and super paramagnetic microspheres labeled pairs, selected 1 pairs can be used to detect antibody of Listeria bacteria on Lester; through the establishment of standard curve, increasing the detection sensitivity of bacteria Lester superparamagnetic magnetic immunochromatography card is about 104CFU/m L is determined, the linear range of 104~108CFU/m L; specificity analysis revealed that the specific detection of Carter is better, although with some non pathogenic Mianyang Lester Lester bacteria disease fungus (L.ivanovii) and Sill List Rand (L.seeligeri) has cross reaction, but still has a very high specificity; in 73 different sources of food samples, the detection results of superparamagnetic immune chromatography and fluorescence PCR method 100% matches. The second part is developed to simultaneously detect board dengue virus and Siniro virus combined detection of superparamagnetic magnetic immunochromatography card. By 8 types of dengue virus antibody, 4 kinds of West Nile virus antibody spray membrane and superparamagnetic microspheres were screened 1 marker pairs, which can be used to the detection of dengue virus and for the detection of West Nile virus antibody; through the establishment of standard curve to determine the detection sensitivity to dengue virus 10ng/m L 10ng/m L~10000ng/m L, the linear range, sensitivity of detection of West Nile virus is 100ng/m L 100ng/m L~10000ng/m L, the linear range for specific detection; The combined detection card detection of dengue virus and Siniro virus partial cross reaction with each other, detection of yellow fever virus and Japanese encephalitis virus has no cross reaction of 100 port inspection; through the actual test samples (25 of which were dengue virus nucleic acid positive samples, 75 negative samples), we found that the accuracy of combined detection card detection dengue virus is 80%, false positive rate 29.3%, false positive rate of detection of West Nile virus for 89.9%. have been established in this study for Listeria List Rand, dengue virus and Siniro virus rapid detection of superparamagnetic immunochromatography, the sensitivity and linear range were higher than that of conventional immunochromatography; detection of Listeria List Randt with specific fluorescent PCR method is simple, fast,; is a new test for reliable. This study successfully screened Lester Listeria bacteria and dengue virus detection The antibody can be used as diagnostic reagents, the development of technology reserve paired antibodies, against Listeria bacteria Lester and dengue virus detection of superparamagnetic immunochromatographic technique has been part of the invention patent and utility model patents, is conducive to the technique further to the disease prevention department, port inspection and quarantine institutions, the promotion and application of the hospital other units, improve the technical capacity of these pathogens. At the beginning of the scene screen

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R446.6

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