第8章 微生物感染的病原學檢查法課件

MedicalMicrobiologyDepartmentofMicrobiology,HMU第8章微生物感染的病原學檢查法第一篇微生物學基礎第8章微生物感染的病原學檢查法LaboratoryDiagnosisZhao-HuaZhong,Prof.第8章微生物感染的病原學檢查法教學大綱掌握內(nèi)容細菌細菌感染的微生物學檢查程序直接涂片鏡檢、分離培養(yǎng)和鑒定、生化試驗、血清學試驗及動物實驗病原菌抗原成分、核酸及其他成分的檢測方法血清學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)細胞中的增殖的指標病毒數(shù)量與感染性的測定（TCID50、PFU的原理、應用）病毒成份的檢測（抗原、核酸檢測）病毒抗體檢測常用方法的原理及應用、雙份血清診斷意義熟悉內(nèi)容標本的采集與送檢原則革蘭染色和抗酸染色的原理細菌藥敏試驗真菌感染的微生物學檢查法了解內(nèi)容組織培養(yǎng)；聚合酶鏈反應、蛋白印跡技術的原理和應用第8章微生物感染的病原學檢查法OutlineLaboratorydiagnosisforbacterialinfectionsMorphological--microscopicMolecularSerologicalLaboratorydiagnosisforviralinfectionsMorphological--electronmicroscopicIsolationMolecular:nucleicacidsandproteinsSerologicalLaboratorydiagnosisforfungalinfectionsMorphological--microscopicIsolation第8章微生物感染的病原學檢查法第8章微生物感染的病原學檢查法AssaysMorphologicalassaysLightorelectronmicroscopiesIsolationanddifferentiationSerologicalassaysAntigen-antibodyassaysMolecularassaysMicroorganism’sgene(DNA&RNA)第8章微生物感染的病原學檢查法PrinciplesforSpecimenCollectionSterilemanipulation,avoidcontaminationObtainaspecimen

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

Morphological

ProteinandNucleicacids

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

Mouse

GuineaPig

RabbitDogMonkeyInoculationroutesIntradermalSubcutaneousIntraperitoneal

Intravenous

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

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

quantitatethenumberofthelivingviralparticles第8章微生物感染的病原學檢查法第8章微生物感染的病原學檢查法Plaque第8章微生物感染的病原學檢查法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檢查法病毒的數(shù)量與感染性測定蝕斑測定：準確滴定病毒感染性的方法蝕斑（Plaque）每個蝕斑是由一個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%組織細胞感染量（TCID50）不能準確地測定感染性病毒顆粒的數(shù)量第8章微生物感染的病原學檢查法ProteinsandNucleicAcidsProteins(antigens)Fluoresece-labeled,enzyme-labeled(enzymelinkedimmunosorbentassay,ELISA)andradio-labeledanitbodiesViralDNAorRNAGenechipDNAchip/cDNAMicroarray第8章微生物感染的病原學檢查法Virus-specificAntibodiesNeutralizationtest,NTHemagglutinationinhibitiontest,HIComplementfixation,CFELISAWesternblot第8章微生物感染的病原學檢查法第8章微生物感染的病原學檢查法ELISAforHIVantibody

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