第8章 微生物感染的病原学检查法课件

MedicalMicrobiologyDepartmentofMicrobiology,HMU第8章微生物感染的病原学检查法第一篇微生物学基础第8章微生物感染的病原学检查法LaboratoryDiagnosisZhao-HuaZhong,Prof.第8章微生物感染的病原学检查法教学大纲掌握内容细菌细菌感染的微生物学检查程序直接涂片镜检、分离培养和鉴定、生化试验、血清学试验及动物实验病原菌抗原成分、核酸及其他成分的检测方法血清学诊断原则及常用的方法病毒病毒分离鉴定方法及病毒在培养细胞中的增殖的指标病毒数量与感染性的测定（TCID50、PFU的原理、应用）病毒成份的检测（抗原、核酸检测）病毒抗体检测常用方法的原理及应用、双份血清诊断意义熟悉内容标本的采集与送检原则革兰染色和抗酸染色的原理细菌药敏试验真菌感染的微生物学检查法了解内容组织培养；聚合酶链反应、蛋白印迹技术的原理和应用第8章微生物感染的病原学检查法OutlineLaboratorydiagnosisforbacterialinfectionsMorphological--microscopicMolecularSerologicalLaboratorydiagnosisforviralinfectionsMorphological--electronmicroscopicIsolationMolecular:nucleicacidsandproteinsSerologicalLaboratorydiagnosisforfungalinfectionsMorphological--microscopicIsolation第8章微生物感染的病原学检查法第8章微生物感染的病原学检查法AssaysMorphologicalassaysLightorelectronmicroscopiesIsolationanddifferentiationSerologicalassaysAntigen-antibodyassaysMolecularassaysMicroorganism’sgene(DNA&RNA)第8章微生物感染的病原学检查法PrinciplesforSpecimenCollectionSterilemanipulation,avoidcontaminationObtainaspecimen

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

Morphological

ProteinandNucleicacids

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

Mouse

GuineaPig

RabbitDogMonkeyInoculationroutesIntradermalSubcutaneousIntraperitoneal

Intravenous

Intracranial/intracerebralIntraspinalIntranasalLavage第8章微生物感染的病原学检查法VirulenceTestMedianlethaldose,LD50Medianinfectivedose,ID50ElekPlateDiphtherotoxinCorynebacteriumdiphtheriae

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

quantitatethenumberofthelivingviralparticles第8章微生物感染的病原学检查法第8章微生物感染的病原学检查法Plaque第8章微生物感染的病原学检查法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学检查法病毒的数量与感染性测定蚀斑测定：准确滴定病毒感染性的方法蚀斑（Plaque）每个蚀斑是由一个有感染性病毒颗粒形成，称为蚀斑形成单位（Plaqueformingunit，PFU）。病毒的感染量用PFU/ml表示50%感染量（ID50）或50%组织细胞感染量（TCID50）不能准确地测定感染性病毒颗粒的数量第8章微生物感染的病原学检查法ProteinsandNucleicAcidsProteins(antigens)Fluoresece-labeled,enzyme-labeled(enzymelinkedimmunosorbentassay,ELISA)andradio-labeledanitbodiesViralDNAorRNAGenechipDNAchip/cDNAMicroarray第8章微生物感染的病原学检查法Virus-specificAntibodiesNeutralizationtest,NTHemagglutinationinhibitiontest,HIComplementfixation,CFELISAWesternblot第8章微生物感染的病原学检查法第8章微生物感染的病原学检查法ELISAforHIVantibody

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