登革病毒感染诊断方法的研究

【作者】 杭小同；

【导师】 李德新；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2010， 博士

【摘要】 登革病毒(Dengue virus)是登革热(Dengue fever, DF)、登革出血热(Dengue haemorrhagic fever, DHF)和登革休克综合症(Dengue shock syndromc, DSS)的病原体,其中以登革热最为常见。登革热广泛流行于热带和亚热带地区,是分布最广,发病最多,危害较大的一种传染病。近几十年来,登革病毒的传播方式由具有较长间歇期的间歇性短暂的一种型别登革病毒流行演变为每年多个地区暴发流行并且持续流行多种血清型的登革病毒感染,登革病毒的流行区域呈不断扩大趋势,对非洲、美洲、地中海东部、东南亚及西太平洋等热带和亚热带地区100多个国家的25亿人口构成威胁。我国,具有较高密度伊蚊等蚊媒地区十分广大,每年都至少有数百例病例报告,输入性病例每年在大部分省份都有发生,有效控制登革疫情传播和登革病毒本土化的任务艰巨。实验室诊断,尤其是对登革病毒感染的早期诊断是疾病监测和有效预防控制重要前提。目前有限的商业化抗登革病毒IgM/IgG检测试剂仍然存在敏感性和特异性问题,而且价格昂贵,不适宜我国及周边发展中国家使用,基于核酸的检测方法也存在规范化和标准化的问题,因此,用于登革热检测和监测的有效的病毒核酸和蛋白诊断试剂及标准操作程序已成为我国当前登革热预防控制任务需要迫切解决的问题。本研究主要目,的建立基于病毒核酸的检测方法、基于检测病毒抗原的检测方法和基于病毒特异性抗体的检测方法等。一,建立登革病毒RT-PCR核酸检测竞争性假病毒颗粒型内参照品,规范基于核酸的检测方法。将筛选确定的登革病毒核酸检测靶基因序列间引入与登革病毒无关基因序列构建内参照检测靶靶标,使之与登革病毒检测靶标片段大小明显不同,易于区分,可作为内参照品形式存在,同时,为了方便对内参照品的定量分析,在内参照品检测靶标的下游通过核糖体内部进入位点基因介导的荧光蛋白报告基因,形成内参照品靶标定性定量指示盒。通过慢病毒包装系统,将参照品靶标定性定量指示盒包装成有包膜的假RNA病毒颗粒,靶标下游报告基因的存在,使内参照照品制备过程中的定性指示和初步的定量分析,十分方便。收获假病毒颗粒,定量分析后加入到到待检样本和模拟样本中一起进行评价和应用。结果显示所构建的登革病毒病毒核酸检测竞争性假病毒颗粒型内参照品能够充当RT-PCR法检测不同型别登革病毒的内参照品,与登革病毒十分接近,扩增片段大小和病毒目的扩增片段明显不同,易于区别,能够监测反应的整个过程。本研究所建立的登革病毒核酸检测竞争性假病毒颗粒型内参照系统,容易制备,保存简单,易于改造为多种RNA病毒的核酸检测的内参照。二,建立登革病毒一步法多重实时定量RT-PCR分型检测方法通过对Genbank所能获取的登革病毒全基因序列和部分序列使用Bioedit软件进行比对,每个型别病毒设计3组登革病毒分型TaqMan探针及各型相应的引物,以标定好效价的登革病毒上清进行梯度稀释作为评价检测限的模拟样本。经比较研究确定了最佳探针引物,其对1-4型革病毒上清的检测限分别是0.1TCID50/ML,0.01 TCID50/ML,0.001TCID50/ML,0.001TCID50/ML。特异性分析证明该套探针和引物具有型特异性,各型病毒之间没有交叉反应,对于汉坦病毒和乙脑病毒等其他科属病毒也未出现非特异扩增。三,建立基于重组登革病毒EⅢ蛋白的登革病毒特异性IgM抗体检测方法。血清中登革病毒特异性IgM／IgG检测试剂的敏感性和特异性一直是困扰目前商业化登革病毒特异性抗体检测试剂的难题。本研究中通过将4型登革病毒E蛋白Ⅲ结构的重组交联,形成融合抗原rEⅢ蛋白。在大肠杆菌BL21 (DE3)中成功表达1-4型登革病毒融合rEⅢ蛋白,SDS-PAGE可溶性分析结果显示表达形式为包涵体。包涵体复性采用稀释复性的方法,复性后蛋白依次经金属螯合层析柱和弱阴离子交换柱纯化,SDS-PAGE结果显示目的蛋白有较高的纯度。Western bolt结果显示表达的重组蛋白均能于登革热感染病人血清发生特异性反应,具有良好的抗原性。采用改良过碘酸盐氧化法rEⅢ融合蛋白进行辣根过氧化物酶(HRP)标记,酶标抗原经分子筛纯化后,初步建立了IgM捕获ELISA的诊断方法,通过棋盘法确定最佳的酶标抗原稀释度和血清稀释度后,rEⅢ-ELISA法与panbio公司MAC-ELISA试剂盒检测结果的符合率为91.11%。使用自研方法检测5份乙脑IgM阳性血清、20份流行出血热病人及20份健康人血清结果均为阴性,说明自研方法的特异性均较好。更多还原

【Abstract】 Dengue virus (DEN) causes Dengue fever (DF), Dengue hemorrhagic fever (DHF) and Dengue Shock Syndrome (DSS). Dengue fever is a febrile illness. Dengue hemorrhagic fever and Dengue Shock Syndrome often prove fatal. Dengue fever virus infection is an important public health issue in the tropics and subtropics. There are still some problems in the control and prevention of Dengue virus infections. By now, no safe and efficient vaccine has been licensed. There is an increasing need for rapid early diagnosis. This study developed diagnostic methods for Dengue infec-tions from aspects of serology and molecular biology.1. Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus.The internal controls target gene were obtained by insertion of a 180bp non-related DNA fragment into RT-PCT detection targeted gene of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus. 2. Real-Time One-Step mutiplex RT-PCR with TaqMan probe for genetyping of dengue virusEarly detection of Dengue infection, especially quantitative and genetyping diagnosis, is very important for clinical treatment of patients and prevention of disease dissemination. Real-Time RT-PCR can be a good method to meet the request.In this study, complete and partial sequences of four serotypes of Dengue virus were loaded from Genbank and multiple alignment were made by the software of Bioedit.12 set of serotype specific TaqMan probes and primer pairs were designed for the real-time genotyping assay..The detection limit of each assay can be 0.1TCID50/ML,0.01TCID50/ML,0.001TCID50/ML,0.001TCID50/ML for the dengue virus type 1-4 respectively. All the four tests are genotype specific and there are also no non-specific amplification for other viruses such as hantaviruses and measles virus.3. As a simple and sensitive method, IgM capture ELISA (MAC-ELISA) is recommended by World Health Organization in the early serological diagnosis. The rE3 proteins were expressed as insoluble proteins in the inclusion bodies as shown by SDS-PAGE. After dilution renaturation, the recombinant proteins were purified by metal chelating and weak anion-exchange chromatography, western bolt results showed that those proteins can react with patient serum specifically.The rEIII fusion protein were conjugated with HRP, then purified by the molecular sieve. rEIII-ELISA methods which use rEIII fusion protein as detection antigen. were established, Commercial panbio MAC-ELISA were used as reference test, The sensitivity, specificity of rEIII-ELISA method are 83.3%,99.5%, The agreement rates of rEIII-ELISA with the commercial panbio MAC-ELISA kit were 91.11%.In addition,5 serologic specimens from Japanese encephalitis virus infected patients,20 serologic specimens from epidemic hemorrhagic fever patients’sera and 20 serologic specimens from healthy persons were tested by self-developed MAC-ELISA methods, and all the results were negative. In conclusion, The self-developed MAC-ELISA methods could be applied in early serodiagnosis of Dengue virus infections to detect virus-specific IgM.更多还原