马尔堡病毒的实时荧光RT-PCR检测方法研究

目的建立马尔堡病毒的实时荧光RT-PCR检测方法。方法人工合成马尔堡病毒特异性核酸序列作为阳性对照模板,设计实时荧光RT-PCR引物、探针并构建反应体系,对反应条件进行优化,验证该方法的特异性、灵敏度。结果建立的马尔堡病毒实时荧光RT-PCR检测方法对马尔堡病毒核酸检测有高度特异性,与1型～4型登革病毒、日本脑炎病毒和基孔肯雅病毒均无交叉反应,检测灵敏度为102拷贝/反应。结论该方法灵敏度高、特异性强,适用于对马尔堡病毒的快速检验。     

苏丹型埃博拉病毒实时荧光PCR方法的建立

目的建立苏丹型埃博拉病毒实时荧光PCR检测方法,为埃博拉病毒分型检测提供技术储备。方法比较15株埃博拉病毒基因序列的保守性和特异性,设计3对引物和探针用于苏丹型埃博拉病毒核酸检测。使用不同浓度的苏丹型埃博拉病毒RNA对照进行检测,确定最佳的引物探针组合、引物探针用量和Mg~(2+)用量,建立苏丹型埃博拉病毒实时荧光PCR方法。对建立的方法进行灵敏度、特异性和精密度分析,并用于广东口岸非洲入境发热人员血清样本的筛查。结果经筛选,设计的引物、探针Ebv-S-2F/Ebv-S-2R/Ebv-S-2P对1.6×10~2～1.6×10~5拷贝/ml的阳性对照检出率为100.0%,是最佳的引物探针组合。优化后的实时荧光PCR体系引物和探针终浓度分别为400 nmol/L和80 nmol/L,Mg~(2+)终浓度为2 mmol/L。灵敏度为80拷贝/ml,重复性好,特异性强,对扎伊尔型、科特迪瓦型、本迪布焦型和莱斯顿型埃博拉病毒阳性对照,马尔堡病毒阳性对照、登革病毒、黄热病毒、基孔肯雅病毒、寨卡病毒等检测结果均为阴性。结论建立的苏丹型埃博拉病毒实时荧光PCR检测方法灵敏度高,特异性强,重复性好,可用于口岸输入性埃博拉出血热病例的早期检测和分型鉴别。     

科特迪瓦型埃博拉病毒实时荧光PCR方法的建立

目的建立科特迪瓦型埃博拉病毒实时荧光PCR检测方法,并在广东口岸进行应用。方法分析埃博拉病毒基因组的同源性,设计3组引物和探针用于科特迪瓦型埃博拉病毒核酸检测。对不同浓度的科特迪瓦型埃博拉病毒体外转录RNA进行检测,确定最佳的引物和探针组合、用量,以及Mg~(2+)用量,建立科特迪瓦型埃博拉病毒实时荧光PCR方法。对建立的方法进行灵敏度、特异性和灵敏度分析,并用于广东口岸非洲入境发热人员血清样本的筛查。结果经筛选,引物Ebv-C-3F/Ebv-C-3R和探针Ebv-C-3P对10~2~10~5拷贝/ml的阳性质粒的检出率为100.0%,是最佳的引物和探针组合。优化后的实时荧光PCR体系中引物和探针终浓度分别为400 nmol/L和80 nmol/L,Mg~(2+)终浓度为2 mmol/L。方法的灵敏度为50拷贝/ml,重复性好、特异性强,对扎伊尔型、苏丹型、本迪布焦型和莱斯顿型埃博拉病毒阳性对照样本、马尔堡病毒阳性对照样本和广东口岸非洲发热入境人员血清样本检测均为阴性。结论建立的科特迪瓦型埃博拉病毒实时荧光PCR检测方法灵敏度高,特异性强,重复性好,对于境外输入性埃博拉病毒病病例的早期诊断和分型鉴别具有重要意义。     

双重荧光RT-PCR检测4种致病性亚型埃博拉病毒方法的建立

目的建立可同时检测苏丹、扎伊尔、本迪布焦和科特迪瓦4种致病性亚型埃博拉病毒的双重荧光RT-PCR检测方法。方法根据4种致病性亚型埃博拉病毒的核蛋白NP基因保守序列,针对苏丹型/扎伊尔型病毒以及针对本迪布焦型/科特迪瓦型病毒,相应设计2套引物和探针。以体外转录的4种亚型埃博拉病毒RNA为模板,进行条件的优化以及方法特异性、灵敏度、重复性试验,建立双重荧光RT-PCR检测方法。结果新建立的双重荧光RT-PCR方法检测只对4种埃博拉病毒阳性对照RNA模板有特异性扩增,与肾综合征出血热病毒、登革病毒、黄热病毒、西尼罗病毒、日本脑炎病毒、基孔肯雅病毒均无交叉反应,2套引物和探针的检测灵敏度均可达到最低50拷贝/μL。苏丹型和本迪布焦型埃博拉病毒阳性对照RNA模板的4种稀释度（2×108、2×106、2×104、2×102拷贝/μL）重复检测3次均有较好的重复性。结论建立的方法能同时检测上述4种亚型埃博拉病毒,灵敏度高,特异性强,可用于埃博拉病毒疑似病例的检测。     

基于新型悬浮芯片的13种/型经呼吸道感染病毒高通量检测方法

目的基于目标特异性引物延伸(TSPE)技术的新型基因悬浮芯片,建立高通量、快速、准确、可同时检测多种呼吸道病原的方法。方法针对流感病毒A、B型,副流感病毒1、3型,冠状病毒SARS-Co V、Co V-NL63、呼吸道合胞病毒A、B型,腺病毒B、E型,鼻病毒、人偏肺病毒、肠道病毒共13种/型经呼吸道感染病毒的特异性基因序列,设计13对引物和相应的特异性探针,经多重PCR扩增及TSPE反应,生物素标记对应基因片段,标记的片段与相应微球结合,悬浮芯片扫描仪检测。结果悬浮芯片检测体系能正确检测13种/型经呼吸道传播的病毒及其亚型,各探针间无交叉反应,特异性好、灵敏度高。结论建立了高通量、快速、准确的可同时检测多种呼吸道病原的技术平台,为突发呼吸道传染病的鉴定和检测提供技术储备,有利于及时应对传染病疫情。     

致命的马尔堡病毒

去年10月，一种比埃博拉病毒更为“猖獗”的病毒在非洲国家安哥拉引起一片恐慌。患者最初会出现高烧、恶心、呕吐、腹泻、头痛、肌肉疼痛等症状，5～7天后会出现严重的出血症状，病死率高达90％以上。现已明确该病是由一种叫马尔堡病毒引起的出血热。世界卫生组织发出警报称：“马尔堡病毒(又名青猴病毒)是迄今为止最具有致命性的病毒。”     

最可怕的病毒

迄今为止,世界上最可怕的生物病毒是什么？ “可怕”的程度或许可以对应为生物性危害等级.美国的疾病管制中心（CDC）将生物性危害分为四个等级,从一级到四级危害性依次增加.像引起水痘的带状疱疹病毒属于一级;乙肝病毒、流感病毒属于二级;天花等属于三级;四级,也就是危害最大的一级,主要包括埃博拉病毒和马尔堡病毒等. 人类往往对生物危害四级的病毒无能为力,比如埃博拉病毒,具有50％ ～ 90％的致死率,致死原因主要为中风、心肌梗塞、低血容量性休克或多发性器官衰竭.     

多种病毒集合检测芯片的研制及其在临床血清标本检测中的初步应用

目的 研制黄热病、西尼罗热、登革热、基孔肯雅热、埃博拉出血热、马尔堡出血热、拉沙热等多种病毒性传染病集合检测基因芯片,并建立一种适用于直接检测核酸含量较低临床血清标本的新型芯片靶基因扩增标记方法.方法 设计并筛选出上述病毒70mer寡核苷酸特异探针各20条,打印于同一基因芯片上；以phi29 DNA聚合酶结合带标签序列的随机引物进行临床标本中病毒全基因组扩增,再以Cy3荧光染料标记的标签序列引物进行PCR随机扩增标记,标记产物用多种病毒芯片进行杂交检测.结果 检测1～4型登革热、基孔肯雅热病例临床血清标本,结果显示该方法准确、特异、敏感；检测西尼罗热、黄热病、埃博拉出血热、马尔堡出血热、拉沙热等病毒核酸的模拟血清标本,同样得到特异的阳性杂交信号,与预期结果一致.结论 本研究建立的多种病毒集合检测基因芯片及其靶基因扩增标记方法,可直接应用于血清标本中上述病毒核酸的检测.     

埃博拉出血热与埃博拉病毒

埃博拉出血热(EHF)是由埃博拉病毒(Ebola Virus,EBO)感染引起的疾病。埃博拉(Ebola)与马尔堡(Marburg)是丝状病毒科仅有的两个属,它们都能导致高致死率和高传染性的出血热病,属于生物安全4级病原因子(危险性最高),人类对他们的了解至今还非常少,本文就目前对埃博拉出血热及埃     

扎伊尔型埃博拉病毒实时荧光定量PCR方法的建立

目的 建立扎伊尔型埃博拉病毒实时荧光定量PCR快速检测技术。方法 人工合成扎伊尔型埃博拉病毒包膜糖蛋白GP基因上1 969～2 113 bp的保守特异片段,克隆到pUC57重组质粒中,构建阳性模板;以103～109拷贝数的重组质粒样品共7组作为标准品,制作标准曲线;设计上游引物5' - GAACCACATGATTGGACCAAGA - 3'、下游引物5' - TAACTCCAATACCTGCCGGTATC - 3'及TaqMan探针FAM 5' - TTGTTGATAAAACCCTTCCGGACCAGG - 3' BHQ1,采用普通PCR和实时荧光定量PCR,检测其特异性和灵敏度。结果 克隆出含GP 1 969～2 113 bp片段的阳性重组质粒pUC57 - GP,其浓度为97.75 ng/μl;以pUC57 - GP为标准品,制备标准曲线,拷贝数为103～109 copies/μL有较好线性关系,相关系数R2 = 0.999;能特异性检测扎伊尔型埃博拉病毒,而与I型登革病毒、寨卡病毒无交叉反应,其检测最低拷贝数达3.10×101copies/μl。 结论 建立了扎伊尔型埃博拉病毒荧光定量PCR快速检测技术,具有快速、灵敏、特异、定量检测等特点,可为埃博拉疫情的综合防控提供技术支撑。     

Ecologic and geographic distribution of filovirus disease

We used ecologic niche modeling of outbreaks and sporadic cases of filovirus-associated hemorrhagic fever (HF) to provide a large-scale perspective on the geographic and ecologic distributions of Ebola and Marburg viruses. We predicted that filovirus would occur across the Afrotropics: Ebola HF in the humid rain forests of central and western Africa, and Marburg HF in the drier and more open areas of central and eastern Africa. Most of the predicted geographic extent of Ebola HF appear to have been observed; Marburg HF has the potential to occur farther south and east. Ecologic conditions appropriate for Ebola HF are also present in Southeast Asia and the Philippines, where Ebola Reston is hypothesized to be distributed. This first large-scale ecologic analysis provides a framework for a more informed search for taxa that could constitute the natural reservoir for this virus family.     

Marburg, Ebola and Rift Valley Fever virus antibodies in East African primates

Sera from 464 primates held at four institutes in Kenya were tested by indirect immunofluorescence for the presence of antibodies against Marburg, Ebola, Congo haemorrhagic fever, Rift Valley fever and Lassa viruses. Four of 136 vervet monkeys were positive for Marburg virus antibodies and three of 184 baboons had antibodies against Ebola virus. One baboon was positive for Marburg virus antibodies. Two vervet monkeys, three baboons and one grivet monkey (of 56 tested) had antibodies against Rift Valley fever virus. No Congo or Lassa virus antibodies were detected. A sample of 88 sera of more arboreal primates (Sykes, blue and colobus monkeys) were negative against all five antigens, as were sera from 58 staff members of the institutes who worked with or near the animals.     

Haemorrhagic fever in Gabon. I. Incidence of Lassa,Ebola and Marburg viruses in Haut-Ogooué

A serological enquiry aimed at determining the incidence of infection with Lassa, Ebola and Marburg viruses was conducted on the human population of the region of Haut-Ogooué (Gabon) and on primates.The results, obtained by the indirect immunofluorescence technique, showed that more than 6% of the human population had had contact with Ebola virus but no antibodies against Marburg or Lassa viruses were found.Most sera reacted to an Ebola antigen from a Zairian strain, but showed little or no reaction to an antigen from a Sudanese strain.     

Risk factors for Marburg hemorrhagic fever, Democratic Republic of the Congo

We conducted two antibody surveys to assess risk factors for Marburg hemorrhagic fever in an area of confirmed Marburg virus transmission in the Democratic Republic of the Congo. Questionnaires were administered and serum samples tested for Marburg-specific antibodies by enzyme-linked immunosorbent assay. Fifteen (2%) of 912 participants in a general village cross-sectional antibody survey were positive for Marburg immunoglobulin G antibody. Thirteen (87%) of these 15 were men who worked in the local gold mines. Working as a miner (odds ratio [OR] 13.9, 95% confidence interval [CI] 3.1 to 62.1) and receiving injections (OR 7.4, 95% CI 1.6 to 33.2) were associated with a positive antibody result. All 103 participants in a targeted antibody survey of healthcare workers were antibody negative. Primary transmission of Marburg virus to humans likely occurred via exposure to a still unidentified reservoir in the local mines. Secondary transmission appears to be less common with Marburg virus than with Ebola virus, the other known filovirus.     

Antibodies against haemorrhagic fever viruses in Kenya populations

Human sera from Lodwar (77 sera), Nzoia (841 sera), Masinga (251 sera), Laisamis (174 sera) and the Malindi/Kilifi area (556 sera) in Kenya were tested by indirect immunofluorescence for antibodies against Marburg, Ebola (Zaire and Sudan strains), Congo haemorrhagic fever, Rift Valley fever and Lassa viruses. Antibodies against Ebola virus, particularly the Zaire strain, were detected in all regions and were, over-all, more abundant than antibodies against the other antigens. Ebola and Marburg antibody prevalence rates were highest in the samples from Lodwar and Laisamis, both semi-desert areas. Antibodies against Rift Valley fever virus were also highest in the Lodwar sample followed by Malindi/Kilifi and Laisamis. Congo haemorrhagic fever virus antibodies were rare and no antibodies against Lassa virus were detected in the 1899 sera tested.     

Feasibility of Xpert Ebola Assay in Médecins Sans Frontières Ebola Program,Guinea

Rapid diagnostic methods are essential in control of Ebola outbreaks and lead to timely isolation of cases and improved epidemiologic surveillance. Diagnosis during Ebola outbreaks in West Africa has relied on PCR performed in laboratories outside this region. Because time between sampling and PCR results can be considerable, we assessed the feasibility and added value of using the Xpert Ebola Assay in an Ebola control program in Guinea. A total of 218 samples were collected during diagnosis, treatment, and convalescence of patients. Median time for obtaining results was reduced from 334 min to 165 min. Twenty-six samples were positive for Ebola virus. Xpert cycle thresholds were consistently lower, and 8 (31%) samples were negative by routine PCR. Several logistic and safety issues were identified. We suggest that implementation of the Xpert Ebola Assay under programmatic conditions is feasible and represents a major advance in diagnosis of Ebola virus disease without apparent loss of assay sensitivity.     

8种重大烈性传染病病毒液相芯片检测方法的建立

目的建立基于多重PCR技术结合液相芯片技术同时检测汉坦病毒（HTV）、裂谷热病毒（RVFV）、黄热病毒（YFV）、西尼罗病毒（WNV）、克里米亚-刚果出血热病毒（CCHFV）、拉沙热[下同]病毒（LFV）、埃博拉病毒（EBV）和马尔堡病毒（MBV）的方法。方法建立8种病毒同时扩增的多重PCR反应体系,分别用各种病毒特异的核酸探针偶联不同编码的微球,将获得的PCR产物与偶联核酸探针的微球混合物进行杂交,建立液相芯片检测方法,并对建立的液相芯片检测方法进行灵敏度及特异性检测评价。结果建立的8种重大烈性传染病病毒的液相芯片筛查方法具有较高的特异性和敏感性,能对8种病毒进行特异的检测。灵敏性实验结果表明,RVFV为10 ng/PCR、WNV为1 ng/PCR、EBV为10 ng/PCR、CCHFV为10 pg/PCR、MBV为1 ng/PCR、HTV为100 pg/PCR、LFV为1 ng/PCR、YFV为10 pg/PCR。结论本研究建立的病毒液相芯片筛查方法能快速、敏感、特异地同时检测8种重大烈性传染病病毒,对口岸入境人员是否携带重大烈性传染病病毒的快速筛查具有广阔的应用前景。