临床标本中未知病毒基因芯片检测方法探索

目的探索可用于检测临床标本中未知病毒的基因芯片技术。方法设计合成1～4型登革病毒基因芯片探针,制备成登革病毒基因芯片。提取1～4型登革病毒标准毒株的核酸RNA,以phi29DNA聚合酶结合带标签序列的随机引物进行全基因组扩增,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,标记产物进一步用登革病毒芯片进行杂交检测,随后用广州白云机场出入境检验检疫局国境口岸收集的登革热病人血清标本对新建立的方法进行验证。结果1～4型登革病毒标准毒株培养液提取的RNA,经扩增、标记及芯片杂交检测,相应探针阵列均可检测到明显杂交信号,探针阳性率均达100％;从3份临床登革热病人血清标本中提取的RNA,同样也可得到明显的杂交信号,而背景干扰信号很低,从相应达到100％探针阳性率的探针阵列可判断,3份病人血清标本中分别为含1、2、3型登革病毒。结论该研究新建立的基因芯片方法,可用于检测临床血清标本中的登革病毒,如果设计更多针对不同病原体的特异性芯片探针,该方法还可用于临床标本中一些未知病原体的鉴定。     

6种虫媒病毒微孔膜芯片检测方法的研制与应用

目的研制能同时检测6种口岸重要虫媒病毒的微孔膜芯片。方法针对包括1—4型登革病毒、乙型脑炎病毒、西尼罗病毒、黄热病毒、基孔肯雅病毒和裂谷热病毒等6种虫媒病毒,选择合适的保守基因,设计特异性的PCR引物（5’端标记生物素）和检测探针,通过参数优化建立单管多重RT—PCR扩增体系;然后按每个阵列5X5的格式,并确保点样区域为96孔板的微孔大小,将探针喷点到处理后的尼龙膜上,通过条件优化建立稳定的PCR产物与固化探针的杂交体系;采用碱性磷酸酯酶标记链亲和素和化学显色底物NBT／BCIP来检测特异性的PCR杂交产物。选取2012年1—6月份从口岸输入的疑似登革热发热病例的临床血清标本,提取RNA后,直接采用本研究建立的微孔膜芯片进行未知虫媒病毒的快速检测。结果用1～4型登革病毒、乙型脑炎病毒、西尼罗病毒和基孔肯雅病毒等7种毒株、1种黄热病毒疫苗株和1种裂谷热病毒核酸体外转录的RNA模板验证已建立的微孔膜芯片,获得比较特异和稳定的实验结果。应用该研究建立的方法,从3份疑似登革热发热病例的临床血清标本中检出了1例登革1型病毒和2例登革2型病毒,与实时荧光PCR检测结果相符。结论该研究建立的6种虫媒病毒微孔膜芯片检测方法,具有快速、准确、自动化和高通量等特点,为快速应对口岸输入性发热病例提供了非常有价值的检测手段,也为进一步开发更多指标的病原体检测方法提供良好的示范作用。     

两种出血热病毒新型液相芯片检测体系的建立

目的 建立两种出血热生物恐怖病毒(马尔堡病毒、埃博拉病毒)的新型液相芯片检测方法.方法针对马尔堡病毒、扎伊尔埃博拉病毒的特异性基因序列设计2对引物和相应的TSPE引物.经多重PCR扩增之后、加入连有TAG的TSPE引物特异性识别靶目标,标记有生物素的dCTP加入到延伸序列中,TAG与微球上的anti-TAG互补,SAP...     

黄热病毒与甲型流感病毒及其分型集合检测芯片的初步研制

目的：研制黄热病毒与流感病毒及其分型集合检测芯片。方法：设计并合成60mer寡核苷酸（Oligo）探针用于制备检测芯片。提取黄热病、甲型流感病毒核酸,以限制性显示技术进行扩增标记,完成杂交后对芯片进行扫描和数据分析。结果：Oligo探针与相应的荧光标记样本杂交后,大部分能检测出阳性荧光信号,而空白对照和阴性对照均为阴性信号。结论：寡核苷酸芯片技术可应用于多种病毒的检测及分型,从而为多种病毒感染的早期鉴别诊断提供科学依据。     

大连口岸首例输入性基孔肯雅热病例的实验室检测和确认

目的对大连口岸1名入境发热者的血液样本进行检测和确认,为输入性基孔肯雅热疫情的处置提供技术支撑。方法利用核酸检测方法对入境旅客血液样本进行基孔肯雅病毒、登革病毒、寨卡病毒、黄热病毒及疟原虫等虫媒传染病病原体进行筛查。结果经检测确认,该样本基孔肯雅病毒核酸阳性,特异性引物扩增获得序列与已知基孔肯雅病毒核酸序列同源性达99.61%。结论大连口岸存在基孔肯雅热输入风险,应加强国境口岸基孔肯雅热防控。     

基孔肯亚热、克里米亚-刚果出血热、裂谷热病毒核酸液相芯片检测方法的建立

目的建立基孔肯亚热、克里米亚-刚果出血热、裂谷热病毒核酸液相芯片检测方法。方法建立探针偶联至荧光微球、多重PCR扩增方法、杂交检测方法,并对所建立的液相芯片检测方法的灵敏度和特异性进行评价。结果建立的液相芯片检测方法能对基孔肯亚热、克里米亚-刚果出血热、裂谷热病毒中的任意1种、任意2种或者3种同时进行筛查检测,基孔肯亚热病毒检测的灵敏度为1×104copies/PCR,克里米亚-刚果出血热病毒检测的灵敏度为1×105copies/PCR,裂谷热病毒检测的灵敏度为1×103 copies/PCR。该方法对汉坦病毒、埃博拉病毒、黄热病毒、西尼罗病毒、马尔堡病毒的检测结果均为阴性。结论该方法具有高通量、多重检测、快速、敏感、特异的特点,为基孔肯亚热、克里米亚-刚果出血热、裂谷热病毒的筛查检测提供一种新方法。     

重组酶介导扩增方法快速检测黄热病毒

目的本研究采用重组酶介导的等温核酸扩增方法(RAA),通过使用逆转录酶,建立黄热病毒的一步法等温核酸扩增(RT-RAA)方法。方法根据黄热病毒基因组保守序列设计引物和探针,建立并分析RT-RAA的重复性、特异性、灵敏度;以所建立方法对黄热病毒样本进行检测,同时以基因测序进行验证。结果黄热病毒RT-RAA扩增,体系中加入40 U的逆转录酶扩增效果最佳。该方法检测时间短(20 min),并且灵敏度高,检测下限可达100 copy,与登革病毒、西尼罗病毒、日本乙型脑炎病毒、基孔肯雅热病毒等蚊媒病毒无交叉反应,具有良好的特异性。结论构建的黄热病毒RT-RAA方法具有快速、特异以及灵敏的特点,适应于黄热病毒的口岸快速检测。     

恙螨体内汉坦病毒核酸的基因芯片检测

目的 建立快速、灵敏、特异的汉坦病毒基因芯片诊断方法。方法 根据发表的汉坦病毒属(HV)76-118株和R22株S基因核苷酸序列，设计引物和探针，制备寡核苷酸芯片。用CV3标记核苷和引物，运用不对称PCR技术制备单链荧光标记核苷酸片段，并与芯片上的寡核苷酸探针杂交，荧光扫描仪检测并分析信号。结果 研究制备的基因芯片能够检测汉坦病毒HTN型和SEO型病毒核酸的特异性荧光信号。结论 HV的基因芯片检测具有特异、灵敏、快速的优点。基因芯片的制备和检测技术的建立，可为肾综合征出血热等传染病的诊断和预防提供理想的方法。     

中国淮阳山地区由新蜱传布尼亚病毒引起的出血热

病毒性出血热是一种临床症状表现为急性发热和出血的疾病.病原体为人兽共患及节肢动物携带传播的RNA病毒,包括丝状病毒科(如埃博拉出血热、马尔堡出血热)、沙粒病毒科(拉沙热)、黄病毒科(如登革出血热、黄热病)和布尼亚病毒科(如克里米亚-刚果出血热、肾综合征出血热).     

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。     

Undiagnosed Acute Viral Febrile Illnesses,Sierra Leone

Sierra Leone in West Africa is in a Lassa fever–hyperendemic region that also includes Guinea and Liberia. Each year, suspected Lassa fever cases result in submission of ≈500–700 samples to the Kenema Government Hospital Lassa Diagnostic Laboratory in eastern Sierra Leone. Generally only 30%–40% of samples tested are positive for Lassa virus (LASV) antigen and/or LASV-specific IgM; thus, 60%–70% of these patients have acute diseases of unknown origin. To investigate what other arthropod-borne and hemorrhagic fever viral diseases might cause serious illness in this region and mimic Lassa fever, we tested patient serum samples that were negative for malaria parasites and LASV. Using IgM-capture ELISAs, we evaluated samples for antibodies to arthropod-borne and other hemorrhagic fever viruses. Approximately 25% of LASV-negative patients had IgM to dengue, West Nile, yellow fever, Rift Valley fever, chikungunya, Ebola, and Marburg viruses but not to Crimean-Congo hemorrhagic fever virus.     

Arbovirus infections and viral haemorrhagic fevers in Uganda: a serological survey in Karamoja district, 1984

Sera collected in May 1984 from 132 adult residents of Karamoja district, Uganda, were examined by haemagglutination inhibition tests for antibodies against selected arboviruses, namely Chikungunya and Semliki Forest alphaviruses (Togaviridae); dengue type 2, Wesselsbron, West Nile, yellow fever and Zika flaviviruses (Flaviviridae); Bunyamwera, Ilesha and Tahyna bunyaviruses (Bunyaviridae); and Sicilian sandfly fever phlebovirus (Bunyaviridae); and by immunofluorescence tests against certain haemorrhagic fever viruses, Lassa fever arenavirus (Arenaviridae), Ebola-Sudan, Ebola-Za?re and Marburg filoviruses (Filoviridae), Crimean-Congo haemorrhagic fever nairovirus and Rift Valley fever phlebovirus (Bunyaviridae). Antibodies against Chikungunya virus were the most prevalent (47%), followed by flavivirus antibodies (16%), which were probably due mainly to West Nile virus. No evidence of yellow fever or dengue virus circulation was observed. A few individuals had antibodies against Crimean-Congo haemorrhagic fever, Lassa, Ebola and Marburg viruses, suggesting that these viruses all circulate in the area.     

Panmicrobial oligonucleotide array for diagnosis of infectious diseases

To facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed GreeneChipPm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. Methods for nucleic acid preparation, random primed PCR amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. Analysis of nasopharyngeal aspirates, blood, urine, and tissue from persons with various infectious diseases confirmed the presence of viruses and bacteria identified by other methods, and implicated Plasmodium falciparum in an unexplained fatal case of hemorrhagic feverlike disease during the Marburg hemorrhagic fever outbreak in Angola in 2004-2005.     

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.     

Antibodies to haemorrhagic fever viruses in Madagascar populations

Sera of 381 adult people from 5 areas in Madagascar were tested by the indirect immunofluorescence method for antibodies against Congo-Crimean haemorrhagic fever and Rift Valley fever viruses (Bunyaviridae), Ebola (strains Zaire and Sudan) and Marburg viruses (Filoviridae), and Lassa virus (Arenaviridae). The highest prevalence rate was that of Ebola virus (4.5%). As no haemorrhagic syndrome has been found associated with this virus, the possible presence of a less pathogenic, antigenically related, strain is discussed. The prevalences of Congo-Crimean haemorrhagic fever and Rift Valley viruses were very low, despite previous viral isolations from potential vectors. No serum reacted against Lassa or Marburg antigens. The results are analysed in the light of the geographical and bioecological characteristics of Madagascar, which is a true 'microcontinent' very different from the African mainland.     

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。