病原学检测法

第一篇微生物学(wēishēnɡwùxué)基础第8章微生物感染(gǎnrǎn)的病原学检查法LaboratoryDiagnosis第一页，共六十二页。编辑课件教学大纲(jiàoxuédàɡānɡ)掌握内容(nèiróng)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二页，共六十二页。编辑课件OutlineLaboratorydiagnosisforbacterialinfectionsMorphological--microscopicMolecularSerologicalLaboratorydiagnosisforviralinfectionsMorphological--electronmicroscopicIsolationMolecular:nucleicacidsandproteinsSerologicalLaboratorydiagnosisforfungalinfectionsMorphological--microscopicIsolation第三页，共六十二页。编辑课件第四页，共六十二页。编辑课件AssaysMorphologicalassaysLightorelectronmicroscopiesIsolationanddifferentiationSerologicalassaysAntigen-antibodyassaysMolecularassaysMicroorganism’sgene(DNA&RNA)第五页，共六十二页。编辑课件PrinciplesforSpecimenCollectionSterilemanipulation,avoidcontaminationObtainaspecimen

fromtheinfectedsiteaccordingtothediseasephasebeforeadministratingantibioticsDoubleseraforantibodydetections,acuteandrecoveryphaseeachPositiveifthetiterofthelaterspecimenisabove4XtothefirstspecimenTransport,andstorecorrectlyassoonaspossibleStoreat-4oCformostbacterialspecimens,butkeepwarmforNeisseriameningitidisandNeisseriagonorrhoeaeStoreat-4oC~-70oCforviralspecimens第六页，共六十二页。编辑课件LaboratoryDiagnosisforBacterialInfections

Morphological

ProteinandNucleicacids

Antibody第七页，共六十二页。编辑课件BacteriologicalDiagnosisIdentifyingtheorganismMorphologicalIsolation&cultureBiochemicalreactionSerologicalassaysPathogenesis&antibioticssusceptibilityAnimalexperimentVirulencetestantibioticssusceptibility第八页，共六十二页。编辑课件MorphologicalNon-stainedmicroscopicobservationDark-fieldmicroscopyObservingthemovementoflivebacteriaStainedmicroscopicobservationsGramstainAcid-faststainFluorescencestain第九页，共六十二页。编辑课件第十页，共六十二页。编辑课件第十一页，共六十二页。编辑课件第十二页，共六十二页。编辑课件第十三页，共六十二页。编辑课件第十四页，共六十二页。编辑课件第十五页，共六十二页。编辑课件Isolation&Culture:ColonySizeShapeColorSurfacefeaturesSmooth-RoughTransparencyHemolysis第十六页，共六十二页。编辑课件BiochemicalReactions

SugarFermentationH2STestCitrateutilization第十七页，共六十二页。编辑课件SerologicalAssaysDetectionantibodyinthepatient’sserumAcurrentinfectionshouldbeIgMpositiveA4-foldorgreaterriseonantibodytiterbetweentheacuteserumsampleandtheconvalescentserumsampleMajordrawbacksAsingleIgGantibodytiterisdifficulttointerpretbecauseitisunclearwhetheritrepresentsacurrentorapreviousinfectiontheconvalescentsampleisusuallytaken10-14daysaftertheacutesample.Bythistime,thepatienthasoftenrecoveredandthediagnosisbecomesaretrospectiveoneSomeexceptionsIncertaindiseases,asingletiterofsufficientmagnitudecanbeusedaspresumptiveevidenceofacurrentinfection第十八页，共六十二页。编辑课件AnimalexperimentAnimals

Mouse

GuineaPig

RabbitDogMonkeyInoculationroutesIntradermalSubcutaneousIntraperitoneal

Intravenous

Intracranial/intracerebralIntraspinalIntranasalLavage第十九页，共六十二页。编辑课件VirulenceTestMedianlethaldose,LD50Medianinfectivedose,ID50ElekPlateDiphtherotoxinCorynebacteriumdiphtheriae

EnterotoxinenterotoxigenicE.coli第二十页，共六十二页。编辑课件AntibioticSusceptibilityTestMethod第二十一页，共六十二页。编辑课件MIC&MBCMinimumInhibitoryConcentration，MICMinimumBactericidalConcentration，MBCBactericidaldrugsusuallyhaveanMBCequalorverysimilartotheMICBacteriostaticdrugsusuallyhaveanMBCsignificantlyhigherthantheMIC第二十二页，共六十二页。编辑课件第二十三页，共六十二页。编辑课件BacterialProteins,DNA&RNAAntigens(Proteins)KnownantibodiesAgglutination,coagulation,precipitation,ELISA,Immunofluorescence,radioimmunoassayDNA&RNAPCRNucleicacidhybridization第二十四页，共六十二页。编辑课件DetectionofBacterialAntigensPrecipitationtestCoagglutinationtestImmunoflorecence,IFMcAbtechnique第二十五页，共六十二页。编辑课件第二十六页，共六十二页。编辑课件第二十七页，共六十二页。编辑课件第二十八页，共六十二页。编辑课件BacterialDNA&RNAPCRDNAprobe&hybridizationDNAhybridization(southernblot)PlasmidfingerprintanalysisRestrictmultimorphologicanalysis第二十九页，共六十二页。编辑课件第三十页，共六十二页。编辑课件第三十一页，共六十二页。编辑课件PCR第三十二页，共六十二页。编辑课件第三十三页，共六十二页。编辑课件GeneChip/microarray第三十四页，共六十二页。编辑课件DiagnosisStrategyforBacterialInfection第三十五页，共六十二页。编辑课件LaboratoryDiagnosisforViralInfection

MorphologicalIsolation&CultureProtein&NucleicacidsSerological第三十六页，共六十二页。编辑课件MorphologicalElectronmicroscopy,EMImmunoelectronmicroscopy,IEMLightMicroscopyInclusionbodyNegribody第三十七页，共六十二页。编辑课件第三十八页，共六十二页。编辑课件IsolationandCultureAnimalinoculationChickeneggculture:9~14daysTissuecultureOrgancultureTissuecultureCellculture第三十九页，共六十二页。编辑课件Cellculture第四十页，共六十二页。编辑课件第四十一页，共六十二页。编辑课件TypeofCulturedCellsPrimarycellculturePassage2-3generationsDiploidcellculture2-foldchromosomes,Passage50~100generations,safe,canbeusedforvaccinemanufactureContinuouscelllinecultureMutantdiploidcells,mostlytumorcells,easytopassageandmaintainNotsensitiveasdiploidcells,mayhaveendogenousviruses,cannotbeusedforvaccinemanufacture第四十二页，共六十二页。编辑课件IndicationsofViralPropagationCytopathiceffect,CPEHemadsorptionHemagglutinationViralinterferenceNeutralizationtest,NT第四十三页，共六十二页。编辑课件CytopathicEffect,CPE第四十四页，共六十二页。编辑课件ViralQuantityDeterminationTCID5050%tissuecultureinfectiousdoseDeterminetheinfectivityoftheexaminedvirusesPFUPlaqueformationpfu/mlPrecisely

quantitatethenumberofthelivingviralparticles第四十五页，共六十二页。编辑课件第四十六页，共六十二页。编辑课件Plaque第四十七页，共六十二页。编辑课件Differencesbetweenviralparticle(VP),plaqueformationunit(PFU)Viralparticles(VPs)

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

representsthenumberofinfectiousorliveviruses.ItreflectstheamountofworkingvirusesinthepreparationFormostviruspreps,theVP/PFUratiois20:1to50:1Whichonebetterreflectstheamountofactivevirusused?UsingVP(viralparticles)astheunitwillresultinsignificantvariationsintheamountofactuallivevirusesused,andusingIFUorPFUastheviralunitwillgivemoreconsistentoutcomes.第四十八页，共六十二页。编辑课件病毒(bìngdú)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(chēnɡwéi)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四十九页，共六十二页。编辑课件ProteinsandNucleicAcidsProteins(antigens)Fluoresece-labeled,enzyme-labeled(enzymelinkedimmunosorbentassay,ELISA)andradio-labeledanitbodiesViralDNAorRNAGenechipDNAchip/cDNAMicroarray第五十页，共六十二页。编辑课件Virus-specificAntibodiesNeutralizationtest,NTHemagglutinationinhibitiontest,HIComplementfixation,CFELISAWesternblot第五十一页，共六十二页。编辑课件第五十二页，共六十二页。编辑课件ELISAforHIVantibody

MicroplateELISAforHIVantibody:colouredwellsindicatereactivity第五十三页，共六十二页。编辑课件第五十四页，共六十二页。编辑课件DiagnosisStrategyforViralInfection第五十五页，共六十二页。编辑课件RapidDiagnosisforViralInfectionMorphologicalEM&IEM,occasionallyLightMicroscopy:inclusionbodyViralantigensImmunofluorescenceRadioimmunoassaysELISASpecificantibodies:IgMNucleicacids:PCR第五十六页，共六十二页。编辑课件LaboratoryDiagnosisforFungalinfectionDirectmicroscopicCultureSerologicalNucleicacids第五十七页，共六十二页。编辑课件MicroscopicObservationSamplesSputum,skinscrapings,nail,hair,lungbiopsy,etc.MethodTreatedwith10%KOHtodissolvetissuematerial