第8章 微生物感染的病原學(xué)檢查法課件

MedicalMicrobiologyDepartmentofMicrobiology,HMU第8章微生物感染的病原學(xué)檢查法第一篇微生物學(xué)基礎(chǔ)第8章微生物感染的病原學(xué)檢查法LaboratoryDiagnosisZhao-HuaZhong,Prof.第8章微生物感染的病原學(xué)檢查法教學(xué)大綱掌握內(nèi)容細(xì)菌細(xì)菌感染的微生物學(xué)檢查程序直接涂片鏡檢、分離培養(yǎng)和鑒定、生化試驗(yàn)、血清學(xué)試驗(yàn)及動(dòng)物實(shí)驗(yàn)病原菌抗原成分、核酸及其他成分的檢測(cè)方法血清學(xué)診斷原則及常用的方法病毒病毒分離鑒定方法及病毒在培養(yǎng)細(xì)胞中的增殖的指標(biāo)病毒數(shù)量與感染性的測(cè)定（TCID50、PFU的原理、應(yīng)用）病毒成份的檢測(cè)（抗原、核酸檢測(cè)）病毒抗體檢測(cè)常用方法的原理及應(yīng)用、雙份血清診斷意義熟悉內(nèi)容標(biāo)本的采集與送檢原則革蘭染色和抗酸染色的原理細(xì)菌藥敏試驗(yàn)真菌感染的微生物學(xué)檢查法了解內(nèi)容組織培養(yǎng)；聚合酶鏈反應(yīng)、蛋白印跡技術(shù)的原理和應(yīng)用第8章微生物感染的病原學(xué)檢查法OutlineLaboratorydiagnosisforbacterialinfectionsMorphological--microscopicMolecularSerologicalLaboratorydiagnosisforviralinfectionsMorphological--electronmicroscopicIsolationMolecular:nucleicacidsandproteinsSerologicalLaboratorydiagnosisforfungalinfectionsMorphological--microscopicIsolation第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法AssaysMorphologicalassaysLightorelectronmicroscopiesIsolationanddifferentiationSerologicalassaysAntigen-antibodyassaysMolecularassaysMicroorganism’sgene(DNA&RNA)第8章微生物感染的病原學(xué)檢查法PrinciplesforSpecimenCollectionSterilemanipulation,avoidcontaminationObtainaspecimen

fromtheinfectedsiteaccordingtothediseasephasebeforeadministratingantibioticsDoubleseraforantibodydetections,acuteandrecoveryphaseeachPositiveifthetiterofthelaterspecimenisabove4XtothefirstspecimenTransport,andstorecorrectlyassoonaspossibleStoreat-4oCformostbacterialspecimens,butkeepwarmforNeisseriameningitidisandNeisseriagonorrhoeaeStoreat-4oC~-70oCforviralspecimens第8章微生物感染的病原學(xué)檢查法LaboratoryDiagnosisforBacterialInfections

Morphological

ProteinandNucleicacids

Antibody第8章微生物感染的病原學(xué)檢查法BacteriologicalDiagnosisIdentifyingtheorganismMorphologicalIsolation&cultureBiochemicalreactionSerologicalassaysPathogenesis&antibioticssusceptibilityAnimalexperimentVirulencetestantibioticssusceptibility第8章微生物感染的病原學(xué)檢查法MorphologicalNon-stainedmicroscopicobservationDark-fieldmicroscopyObservingthemovementoflivebacteriaStainedmicroscopicobservationsGramstainAcid-faststainFluorescencestain第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法Isolation&Culture:ColonySizeShapeColorSurfacefeaturesSmooth-RoughTransparencyHemolysis第8章微生物感染的病原學(xué)檢查法BiochemicalReactionsSugarFermentationH2STestCitrateutilization第8章微生物感染的病原學(xué)檢查法SerologicalAssaysDetectionantibodyinthepatient’sserumAcurrentinfectionshouldbeIgMpositiveA4-foldorgreaterriseonantibodytiterbetweentheacuteserumsampleandtheconvalescentserumsampleMajordrawbacksAsingleIgGantibodytiterisdifficulttointerpretbecauseitisunclearwhetheritrepresentsacurrentorapreviousinfectiontheconvalescentsampleisusuallytaken10-14daysaftertheacutesample.Bythistime,thepatienthasoftenrecoveredandthediagnosisbecomesaretrospectiveoneSomeexceptionsIncertaindiseases,asingletiterofsufficientmagnitudecanbeusedaspresumptiveevidenceofacurrentinfection第8章微生物感染的病原學(xué)檢查法AnimalexperimentAnimals

Mouse

GuineaPig

RabbitDogMonkeyInoculationroutesIntradermalSubcutaneousIntraperitoneal

Intravenous

Intracranial/intracerebralIntraspinalIntranasalLavage第8章微生物感染的病原學(xué)檢查法VirulenceTestMedianlethaldose,LD50Medianinfectivedose,ID50ElekPlateDiphtherotoxinCorynebacteriumdiphtheriae

EnterotoxinenterotoxigenicE.coli第8章微生物感染的病原學(xué)檢查法AntibioticSusceptibilityTestMethod第8章微生物感染的病原學(xué)檢查法MIC&MBCMinimumInhibitoryConcentration，MICMinimumBactericidalConcentration，MBCBactericidaldrugsusuallyhaveanMBCequalorverysimilartotheMICBacteriostaticdrugsusuallyhaveanMBCsignificantlyhigherthantheMIC第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法BacterialProteins,DNA&RNAAntigens(Proteins)KnownantibodiesAgglutination,coagulation,precipitation,ELISA,Immunofluorescence,radioimmunoassayDNA&RNAPCRNucleicacidhybridization第8章微生物感染的病原學(xué)檢查法DetectionofBacterialAntigensPrecipitationtestCoagglutinationtestImmunoflorecence,IFMcAbtechnique第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法BacterialDNA&RNAPCRDNAprobe&hybridizationDNAhybridization(southernblot)PlasmidfingerprintanalysisRestrictmultimorphologicanalysis第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法PCR第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法GeneChip/microarray第8章微生物感染的病原學(xué)檢查法DiagnosisStrategyforBacterialInfection第8章微生物感染的病原學(xué)檢查法LaboratoryDiagnosisforViralInfectionMorphologicalIsolation&CultureProtein&NucleicacidsSerological第8章微生物感染的病原學(xué)檢查法MorphologicalElectronmicroscopy,EMImmunoelectronmicroscopy,IEMLightMicroscopyInclusionbodyNegribody第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法IsolationandCultureAnimalinoculationChickeneggculture:9~14daysTissuecultureOrgancultureTissuecultureCellculture第8章微生物感染的病原學(xué)檢查法Cellculture第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法TypeofCulturedCellsPrimarycellculturePassage2-3generationsDiploidcellculture2-foldchromosomes,Passage50~100generations,safe,canbeusedforvaccinemanufactureContinuouscelllinecultureMutantdiploidcells,mostlytumorcells,easytopassageandmaintainNotsensitiveasdiploidcells,mayhaveendogenousviruses,cannotbeusedforvaccinemanufacture第8章微生物感染的病原學(xué)檢查法IndicationsofViralPropagationCytopathiceffect,CPEHemadsorptionHemagglutinationViralinterferenceNeutralizationtest,NT第8章微生物感染的病原學(xué)檢查法CytopathicEffect,CPE第8章微生物感染的病原學(xué)檢查法ViralQuantityDeterminationTCID5050%tissuecultureinfectiousdoseDeterminetheinfectivityoftheexaminedvirusesPFUPlaqueformationpfu/mlPrecisely

quantitatethenumberofthelivingviralparticles第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法Plaque第8章微生物感染的病原學(xué)檢查法Differencesbetweenviralparticle(VP),plaqueformationunit(PFU)Viralparticles(VPs)

representthetotalnumberofviralparticles(liveanddeadcombined).Duetovariationsinviruspreparationstheratiooflive/deadvariessignificantlyandtherefore,VPdoesnotreflecttheamountofactivevirusinthepreparationPFU(plaqueformationunit)

representsthenumberofinfectiousorliveviruses.ItreflectstheamountofworkingvirusesinthepreparationFormostviruspreps,theVP/PFUratiois20:1to50:1Whichonebetterreflectstheamountofactivevirusused?UsingVP(viralparticles)astheunitwillresultinsignificantvariationsintheamountofactuallivevirusesused,andusingIFUorPFUastheviralunitwillgivemoreconsistentoutcomes.第8章微生物感染的病原學(xué)檢查法病毒的數(shù)量與感染性測(cè)定蝕斑測(cè)定：準(zhǔn)確滴定病毒感染性的方法蝕斑（Plaque）每個(gè)蝕斑是由一個(gè)有感染性病毒顆粒形成，稱為蝕斑形成單位（Plaqueformingunit，PFU）。病毒的感染量用PFU/ml表示50%感染量（ID50）或50%組織細(xì)胞感染量（TCID50）不能準(zhǔn)確地測(cè)定感染性病毒顆粒的數(shù)量第8章微生物感染的病原學(xué)檢查法ProteinsandNucleicAcidsProteins(antigens)Fluoresece-labeled,enzyme-labeled(enzymelinkedimmunosorbentassay,ELISA)andradio-labeledanitbodiesViralDNAorRNAGenechipDNAchip/cDNAMicroarray第8章微生物感染的病原學(xué)檢查法Virus-specificAntibodiesNeutralizationtest,NTHemagglutinationinhibitiontest,HIComplementfixation,CFELISAWesternblot第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法ELISAforHIVantibody

MicroplateELISAforHIVantibody:colouredwellsindicatereactivity第8章微生物感染的病原學(xué)檢查法第8章微生物感染的病原學(xué)檢查法DiagnosisStrategyforViralInfection第8章微生物感染的病原學(xué)檢查法RapidDiagnosisforViralInfectionMorphologicalEM&IEM,occasionallyLightMicroscopy:inclusionbodyViralantigensImmunofluorescenceRadioimmunoassaysELISASpecificantibodies:IgMNucleicacids:PCR第8章微生物感染的病原學(xué)檢查法LaboratoryDiagnosisforFungalinfectionDirectmicroscopic