1、2023-1-18.2Friend or Foe?Friend or Foe?RotavirusEbola virusBacteriumAlgaeProtozoanVirus2023-1-18.32023-1-18.42023-1-18.52023-1-18.6l大部分人类致命性疾病均是由动物传播，包括现正肆虐全球的禽流感。l每年至少有一种新病原体，包括病毒、细菌、寄生虫、原生动物和真菌等从动物传染给人类。l前所未有的速度产生突变，并传给人类，就如艾滋病、马尔堡出血热、SARS以及现正肆虐全球的禽流感等难抗病毒。l研究人员分辨1407种令人类生病的病原体，发现其中至少800种越过种物界线，由动
2、物传至人类。新发传染病动物传播新发传染病动物传播2023-1-18.72023-1-18.82023-1-18.9人类与传染病较量中的四方面重大人类与传染病较量中的四方面重大突破突破隔离检疫制度的建立;病原微生物的发现;特效药物的出现;疫苗的诞生.2023-1-18.102023-1-18.112023-1-18.122023-1-18.13AdenovirusRotavirus(courtesy of Linda Stannard,University of Cape Town,S.A.)2023-1-18.14Positive immunofluorescence test for rab
3、ies virus antigen.(Source:CDC)(Virology Laboratory,Yale-New Haven Hospital)2023-1-18.15From Principles of Virology,Flint et al ASM pressFrom Principles of Virology,Flint et al ASM press2023-1-18.16From Principles of Virology,Flint et al ASM pressFrom Principles of Virology,Flint et al ASM press2023-
4、1-18.172023-1-18.18genomgenome eprobeprobe3232P P 3535S S 3 3H H 125125I I 生物素生物素 地高辛地高辛 酶酶genomegenomegenomegenomegenomegenomeprobeprobeprobeprobeprobeprobe检测方法：放射自显影检测方法：放射自显影 底物显色底物显色 化学发光化学发光2023-1-18.19斑点杂交（斑点杂交（spot blot hybridization)spot blot hybridization)凝胶电泳印迹转移杂交（凝胶电泳印迹转移杂交（Southern blot
5、 hybridization)Southern blot hybridization)原位杂交（原位杂交（in-situ hybridization)in-situ hybridization)2023-1-18.20聚合酶链反应（聚合酶链反应（PCRPCR）基本步骤和原理基本步骤和原理变性（高温）变性（高温）退火（低温）延伸（适温）延伸（适温）每一循环每一循环模模 板板引物引物引物引物dATP dGTP dTTP dCTP dATP dGTP dTTP dCTP TaqTaq酶酶2023-1-18.21PCRPCR原理和步骤原理和步骤2023-1-18.222023-1-18.242023-
6、1-18.25建立该病原体的鉴定方法动物试验临床实验室诊断流行病学研究等分离到该病原体确定一种疾病病原体的基本步骤确定一种疾病病原体的基本步骤最终确定病原体与疾病的病因学关系2023-1-18.262023-1-18.282023-1-18.29Multiplex PCR-Mass Tag systemMultiplex PCR-Mass Tag system多重 PCR-质谱联用技术2023-1-18.30美国哥伦比亚大学Lipkin 研究组发明了一种基于multiplex PCR-MassTag（多重PCR与质谱联用）的高通量病原体快速筛选技术。优点：由于使用光敏感的分子量标签，增强了mu
7、ltiplex PCR引物设计的适应性和灵活性，使检测通量有大幅度的提高。应用质谱分析PCR产物上的分子量标签克服了凝胶电泳分辨率的局限或荧光染料种类的局限。2023-1-18.311.PCR amplification with conjugated primers1.PCR amplification with conjugated primers90 minutes90 minutes2.PCR purification on filter plate2.PCR purification on filter plate30 minutes30 minutes3.Elution into
8、96-well loading plate3.Elution into 96-well loading platefor mass spectrometer analysisfor mass spectrometer analysis4.Automated sample injection,photocleavage4.Automated sample injection,photocleavageand detectionand detection1:30 minutes/sample1:30 minutes/sampleUltraviolet LightUltraviolet Light5
9、.Identification of pathogen by signal analysis5.Identification of pathogen by signal analysisTHE JEROME L.AND DAWN GREENEINFECTIOUS DISEASE LABORATORYMAILMAN S C H OO L OF PUB L I C H EA L T H Co l u m b i a U n i ver s it yPCR-Mass Tag systemPCR-Mass Tag system2023-1-18.32NUCLEIC ACID CONJUGATED MA
10、SS TAGSNUCLEIC ACID CONJUGATED MASS TAGSTHE JEROME L.AND DAWN GREENEINFECTIOUS DISEASE LABORATORYMAILMAN S C H OO L OF PUB L I C H EA L T H Co l u m b i a U n i ver s it yPhotocleavable linker and spacerPhotocleavable linker and spacerMS sensitivity enhancerMS sensitivity enhancerVariable mass unitV
11、ariable mass unit 2023-1-18.33DETECTION OF 58 DIFFERENT MASS TAGS BY APCI-MSDETECTION OF 58 DIFFERENT MASS TAGS BY APCI-MSTHE JEROME L.AND DAWN GREENEINFECTIOUS DISEASE LABORATORYMAILMAN S C H OO L OF PUB L I C H EA L T H Co l u m b i a U n i ver s it y2023-1-18.34目前，建立在此技术上的有呼吸道病原体检测模块（包括19个病毒和3种细菌
12、），流感病毒亚型检测模块，出血热病原体检测模块，痘疹病毒检测模块和脑炎脑膜炎病原体检测模块。尽管目前已开发的分子量标签只有64个，但根据质谱分析的分子量范围，还可有极大的拓展空间，保证了这一技术具有足够的发展前景和潜力。THE JEROME L.AND DAWN GREENEINFECTIOUS DISEASE LABORATORYMAILMAN S C H OO L OF PUB L I C H EA L T H Co l u m b i a U n i ver s it y2023-1-18.35CurrentCurrent RespiratoryRespiratory PanelPane
13、lInIn ProgressProgressTHE JEROME L.AND DAWN GREENEINFECTIOUS DISEASE LABORATORYMAILMAN S C H OO L OF PUB L I C H EA L T H Co l u m b i a U n i ver s it yEnterovirusEnterovirusAdenovirusAdenovirus2023-1-18.36Current PanelCurrent PanelInIn ProgressProgressTHE JEROME L.AND DAWN GREENEINFECTIOUS DISEASE
14、 LABORATORYMAILMAN S C H OO L OF PUB L I C H EA L T H Co l u m b i a U n i ver s it y2023-1-18.37A AC CB BLabLabNetworkNetworkEpidemiology Epidemiology MicrobiologyMicrobiology research research service service training training infectiousinfectiousagentsagentssamplessamplesclinical dataclinical dat
15、aGLOBAL SURVEILLANCE NETWORK GLOBAL SURVEILLANCE NETWORK Animal modelsAnimal modelsdrugsdrugsvaccinesvaccinesDrosten,HamburgLipkin,New YorkMackenzie,PerthPauli,BerlinPeiris,Hong KongQian,BeijingPerez-Brena,MadridSwanepoel,South AfricaWebster,MemphisWen,Shanghai2023-1-18.39LUMINEX XMAPLUMINEX XMAP技术：
16、多指标并技术：多指标并行检测系统（流行检测系统（流式荧光技术或液式荧光技术或液芯技术）芯技术）技术技术GENOCA GENOCA Templex PCRTemplex PCR技术技术:多指标并多指标并行扩增行扩增2023-1-18.40Super PrimerTemplex PCRTemplex PCR技术原理技术原理2023-1-18.41Templex PCRTemplex PCR技术原理技术原理2023-1-18.422023-1-18.43流式荧光技术（液芯）原理流式荧光技术（液芯）原理XMAPXMAP2023-1-18.44每种微球的地址由两每种微球的地址由两种红色荧光颜料的掺种红色
17、荧光颜料的掺入比例唯一决定入比例唯一决定2023-1-18.45CONH2023-1-18.462023-1-18.472023-1-18.48Luminex 100Luminex 100多功能流式点阵仪多功能流式点阵仪流式荧光检测仪流式荧光检测仪2023-1-18.49 呼吸道感染型11种DNA基因组的病原体2023-1-18.50呼吸道感染型13种RNA基因组病毒型病原体MASATMMASATM液相芯片液相芯片2023-1-18.52 微小的乳胶颗粒（Beads,微球）分别染成不同的荧光色，然后再把针对不同检测物的蛋白（如抗原抗体）以共价方式结合到特定颜色的微球上。应用时，先把针对不同检测
18、物的、用不同颜色编码的微球混合，再加入被检测物（被测物可以是血清中的抗原、抗体、或酶等）。2023-1-18.532023-1-18.542023-1-18.55The Red Laser is The Red Laser is Used to Identify Used to Identify the Bead Codethe Bead CodeThe Green Laser is The Green Laser is Used to Identify the Used to Identify the Reporter SignalReporter Signal2023-1-18.56202
19、3-1-18.572023-1-18.58 MASAMASATMTM和ELISA灵敏度的比较2023-1-18.60基因芯片（基因芯片（Gene ChipGene Chip）何谓基因芯片：何谓基因芯片：基因芯片（或DNA Chip,或Microarray）又称DNA微阵列，是指固定在固相载体上的高密度DNA微点阵。即将大量靶基因或寡核苷酸片段有序地，高密度地（点与点间距一般小于500m）排列在玻璃、硅等载体上，称之为基因芯片。基因芯片的种类：基因芯片的种类：按应用，基因芯片主要可分为3类：1、表达谱基因芯片：主要用于基因功能研究；2、诊断基因芯片：如肝癌诊断芯片、糖尿病诊断芯片、病原体多基因诊
20、断芯片等：3、检测芯片：如商品检疫芯片、病原体检测芯片等。2023-1-18.61Viral Pathogen Custom MicroarrayViral Pathogen Custom Microarray病原体诊断基因芯片2023-1-18.622023-1-18.630.05 CPSMDetect 5 dye molecules in 10um pixel0.1 CPSMDetect 10 dye molecules in 10um pixel2023-1-18.64BackName Your DesignName Your DesignSelect Array FormatSelec
21、t Array Format2023-1-18.65AgrobacteriumAgrobacteriumAnopheles(AG)and Plasmodium(PB).ie.malaria microarrayAnopheles(AG)and Plasmodium(PB).ie.malaria microarrayArchaeon,Halobacterium sp.Archaeon,Halobacterium sp.Bacillus cereus Bacillus cereus Bacterial pathogensBacterial pathogensBifidobacterium long
22、um Bifidobacterium longum Bordetella PertussisBordetella PertussisChlamydophila abortusChlamydophila abortusCulex pipiensCulex pipiensCyanobacterium SynechocystisCyanobacterium SynechocystisE.ColiE.ColiErwinia carotovora atroseptica/Solanum tuberosumErwinia carotovora atroseptica/Solanum tuberosumLa
23、ctobacillus plantarum Lactobacillus plantarum Methylobacterium extorquens AM1Methylobacterium extorquens AM1Plasmodium falciparumPlasmodium falciparumPseudomonas aeruginosaPseudomonas aeruginosaS.aureus;N.meningococcus;E.coliS.aureus;N.meningococcus;E.coliSalmonella typhimuriumSalmonella typhimurium
24、Staphylococcus aureusStaphylococcus aureus2023-1-18.66SARS CoronavirusSARS Coronavirus2023-1-18.67Human Coronavirus OC43Human Coronavirus OC432023-1-18.68Human Coronavirus 229EHuman Coronavirus 229E2023-1-18.69Sample:WNV NY99 Sample:WNV NY99 100,000 RNA copies/ml100,000 RNA copies/ml32/45 top hits a
25、re 32/45 top hits are WNV-specific;WNV-specific;remainder are JEV remainder are JEV group or other group or other flavivirus-genus flavivirus-genus probesprobesCollaboration between the Greene Laboratory of Columbia University,ICTV,Collaboration between the Greene Laboratory of Columbia University,I
26、CTV,Northeast Biodefense Center,and Agilent CorporationNortheast Biodefense Center,and Agilent Corporation2023-1-18.70Sample:SARS-CoVSample:SARS-CoV100,000 RNA copies/ml100,000 RNA copies/mlColumbia(Briese,Lipkin,Liu,Lussier Palacios),ICTV(Bchen-Osmond),Columbia(Briese,Lipkin,Liu,Lussier Palacios),I
27、CTV(Bchen-Osmond),and Agilent(Hirschberg)and Agilent(Hirschberg)251,000 sequence database,1710 vertebrate viruses251,000 sequence database,1710 vertebrate viruses2023-1-18.722023-1-18.732023-1-18.742023-1-18.752023-1-18.762023-1-18.772023-1-18.782023-1-18.792023-1-18.802023-1-18.812023-1-18.822023-1
28、-18.832023-1-18.842023-1-18.852023-1-18.862023-1-18.872023-1-18.882023-1-18.892023-1-18.902023-1-18.912023-1-18.92方法关键环节优点缺点cDNA文库筛选构建文库适用范围广过程繁琐，工作量大，需筛选大量的克隆基于保守序列的PCR引物的选择简便高效局限于检测同源性相近的未知病毒代表性差异分析基于PCR技术的消减杂交非常灵敏不能消除不同mRNA丰度的差异，需多轮杂交，涉及接头连接和去除的效率，技术难抑制性消减杂交差异杂交的cDNA消减重复性高灵敏度低，不易检测低丰度基因SISPA接头与片断
29、的连接、克隆筛选适用范围广易受试剂中的核酸污染，工作量大，需筛选大量的克隆随机PCR法克隆筛选、减少污染适用范围广、简便易受试剂中的核酸污染，工作量大，需筛选大量的克隆芯片芯片的设计通量大成本高，无法克服保守的同源序列及重序对杂交信息的干扰滚环扩增法滚环复制高效只适合于环状病毒2023-1-18.94A Novel Coronavirus AssociatedA Novel Coronavirus Associatedwith Severe Acute Respiratory Syndromewith Severe Acute Respiratory Syndromevirus-isolati
30、on techniquesvirus-isolation techniques electron-microscopical electron-microscopical histologic studieshistologic studiesMolecular and serologic assaysMolecular and serologic assays2023-1-18.952023-1-18.962023-1-18.97Primer pair Primer pair IN-2(+)5GGGTTGGGACTATCCTAAGTGTGA3IN-2(+)5GGGTTGGGACTATCCTA
31、AGTGTGA3IN-4()5TAACACACAACICCATCATCA3IN-4()5TAACACACAACICCATCATCA3conserved regions of open reading frame 1bconserved regions of open reading frame 1bA 405-nucleotide segment2023-1-18.98The Genome sequence of the SARS-associated coronavirus Science 300(5624),1399-1404(2003)Virus isolation(bronchoalv
32、eolar lavage specimen)Viral particles were purified,genetic material(RNA)was extractedRNA was converted to cDNA by random-priming and oligo(dT)primings trategy Size-selected cDNA products were cloned single sequence reads Sequences were assembledRapid amplification of cDNA ends RACE to capture the 5 end of the viral genome.2023-1-18.99病毒性感染样本（咽拭子，痰，肺泡灌洗液，脑脊液）保存活病毒样本核酸抽提、逆转录多重PCR扩增和质谱分析阳性阴性部分样品进行病毒培养阴性阳性部分样品进行基因芯片检测阴性阳性病毒扩增，电子显微镜观察，病毒核酸的分子克隆，序列的比对，进化分析以及基因功能的初步探讨。DNaseI处理-Random PCR克隆扩增DNA，大规模测序，核酸序列比对，进化树分析。不明原因感染的病原体筛查系统不明原因感染的病原体筛查系统/10/29.100NoImage
