2022年醫(yī)學(xué)專題-病原學(xué)檢測法

第一篇微生物學(xué)(wēishēnɡwùxué)基礎(chǔ)第8章微生物感染(gǎnrǎn)的病原學(xué)檢查法LaboratoryDiagnosis第一頁，共六十二頁。教學(xué)大綱(jiàoxuédàɡānɡ)掌握內(nèi)容(nèiróng)細菌細菌感染的微生物學(xué)檢查程序直接涂片鏡檢、分離培養(yǎng)和鑒定、生化試驗、血清學(xué)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(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(yīng)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(yīng)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(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)用第二頁，共六十二頁。OutlineLaboratorydiagnosisforbacterialinfectionsMorphological--microscopicMolecularSerologicalLaboratorydiagnosisforviralinfectionsMorphological--electronmicroscopicIsolationMolecular:nucleicacidsandproteinsSerologicalLaboratorydiagnosisforfungalinfectionsMorphological--microscopicIsolation第三頁，共六十二頁。第四頁，共六十二頁。AssaysMorphologicalassaysLightorelectronmicroscopiesIsolationanddifferentiationSerologicalassaysAntigen-antibodyassaysMolecularassaysMicroorganism’sgene(DNA&RNA)第五頁，共六十二頁。PrinciplesforSpecimenCollectionSterilemanipulation,avoidcontaminationObtainaspecimen

fromtheinfectedsiteaccordingtothediseasephasebeforeadministratingantibioticsDoubleseraforantibodydetections,acuteandrecoveryphaseeachPositiveifthetiterofthelaterspecimenisabove4XtothefirstspecimenTransport,andstorecorrectlyassoonaspossibleStoreat-4oCformostbacterialspecimens,butkeepwarmforNeisseriameningitidisandNeisseriagonorrhoeaeStoreat-4oC~-70oCforviralspecimens第六頁，共六十二頁。LaboratoryDiagnosisforBacterialInfections

Morphological

ProteinandNucleicacids

Antibody第七頁，共六十二頁。BacteriologicalDiagnosisIdentifyingtheorganismMorphologicalIsolation&cultureBiochemicalreactionSerologicalassaysPathogenesis&antibioticssusceptibilityAnimalexperimentVirulencetestantibioticssusceptibility第八頁，共六十二頁。MorphologicalNon-stainedmicroscopicobservationDark-fieldmicroscopyObservingthemovementoflivebacteriaStainedmicroscopicobservationsGramstainAcid-faststainFluorescencestain第九頁，共六十二頁。第十頁，共六十二頁。第十一頁，共六十二頁。第十二頁，共六十二頁。第十三頁，共六十二頁。第十四頁，共六十二頁。第十五頁，共六十二頁。Isolation&Culture:ColonySizeShapeColorSurfacefeaturesSmooth-RoughTransparencyHemolysis第十六頁，共六十二頁。BiochemicalReactions

SugarFermentationH2STestCitrateutilization第十七頁，共六十二頁。SerologicalAssaysDetectionantibodyinthepatient’sserumAcurrentinfectionshouldbeIgMpositiveA4-foldorgreaterriseonantibodytiterbetweentheacuteserumsampleandtheconvalescentserumsampleMajordrawbacksAsingleIgGantibodytiterisdifficulttointerpretbecauseitisunclearwhetheritrepresentsacurrentorapreviousinfectiontheconvalescentsampleisusuallytaken10-14daysaftertheacutesample.Bythistime,thepatienthasoftenrecoveredandthediagnosisbecomesaretrospectiveoneSomeexceptionsIncertaindiseases,asingletiterofsufficientmagnitudecanbeusedaspresumptiveevidenceofacurrentinfection第十八頁，共六十二頁。AnimalexperimentAnimals

Mouse

GuineaPig

RabbitDogMonkeyInoculationroutesIntradermalSubcutaneousIntraperitoneal

Intravenous

Intracranial/intracerebralIntraspinalIntranasalLavage第十九頁，共六十二頁。VirulenceTestMedianlethaldose,LD50Medianinfectivedose,ID50ElekPlateDiphtherotoxinCorynebacteriumdiphtheriae

EnterotoxinenterotoxigenicE.coli第二十頁，共六十二頁。AntibioticSusceptibilityTestMethod第二十一頁，共六十二頁。MIC&MBCMinimumInhibitoryConcentration，MICMinimumBactericidalConcentration，MBCBactericidaldrugsusuallyhaveanMBCequalorverysimilartotheMICBacteriostaticdrugsusuallyhaveanMBCsignificantlyhigherthantheMIC第二十二頁，共六十二頁。第二十三頁，共六十二頁。BacterialProteins,DNA&RNAAntigens(Proteins)KnownantibodiesAgglutination,coagulation,precipitation,ELISA,Immunofluorescence,radioimmunoassayDNA&RNAPCRNucleicacidhybridization第二十四頁，共六十二頁。DetectionofBacterialAntigensPrecipitationtestCoagglutinationtestImmunoflorecence,IFMcAbtechnique第二十五頁，共六十二頁。第二十六頁，共六十二頁。第二十七頁，共六十二頁。第二十八頁，共六十二頁。BacterialDNA&RNAPCRDNAprobe&hybridizationDNAhybridization(southernblot)PlasmidfingerprintanalysisRestrictmultimorphologicanalysis第二十九頁，共六十二頁。第三十頁，共六十二頁。第三十一頁，共六十二頁。PCR第三十二頁，共六十二頁。第三十三頁，共六十二頁。GeneChip/microarray第三十四頁，共六十二頁。DiagnosisStrategyforBacterialInfection第三十五頁，共六十二頁。LaboratoryDiagnosisforViralInfection

MorphologicalIsolation&CultureProtein&NucleicacidsSerological第三十六頁，共六十二頁。MorphologicalElectronmicroscopy,EMImmunoelectronmicroscopy,IEMLightMicroscopyInclusionbodyNegribody第三十七頁，共六十二頁。第三十八頁，共六十二頁。IsolationandCultureAnimalinoculationChickeneggculture:9~14daysTissuecultureOrgancultureTissuecultureCellculture第三十九頁，共六十二頁。Cellculture第四十頁，共六十二頁。第四十一頁，共六十二頁。TypeofCulturedCellsPrimarycellculturePassage2-3generationsDiploidcellculture2-foldchromosomes,Passage50~100generations,safe,canbeusedforvaccinemanufactureContinuouscelllinecultureMutantdiploidcells,mostlytumorcells,easytopassageandmaintainNotsensitiveasdiploidcells,mayhaveendogenousviruses,cannotbeusedforvaccinemanufacture第四十二頁，共六十二頁。IndicationsofViralPropagationCytopathiceffect,CPEHemadsorptionHemagglutinationViralinterferenceNeutralizationtest,NT第四十三頁，共六十二頁。CytopathicEffect,CPE第四十四頁，共六十二頁。ViralQuantityDeterminationTCID5050%tissuecultureinfectiousdoseDeterminetheinfectivityoftheexaminedvirusesPFUPlaqueformationpfu/mlPrecisely

quantitatethenumberofthelivingviralparticles第四十五頁，共六十二頁。第四十六頁，共六十二頁。Plaque第四十七頁，共六十二頁。Differencesbetweenviralparticle(VP),plaqueformationunit(PFU)Viralparticles(VPs)

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

representsthenumberofinfectiousorliveviruses.ItreflectstheamountofworkingvirusesinthepreparationFormostviruspreps,theVP/PFUratiois20:1to50:1Whichonebetterreflectstheamountofactivevirusused?UsingVP(viralparticles)astheunitwillresultinsignificantvariationsintheamountofactuallivevirusesused,andusingIFUorPFUastheviralunitwillgivemoreconsistentoutcomes.第四十八頁，共六十二頁。病毒(bìngdú)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(chēnɡwéi)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)量第四十九頁，共六十二頁。ProteinsandNucleicAcidsProteins(antigens)Fluoresece-labeled,enzyme-labeled(enzymelinkedimmunosorbentassay,ELISA)andradio-labeledanitbodiesViralDNAorRNAGenechipDNAchip/cDNAMicroarray第五十頁，共六十二頁。Virus-specificAntibodiesNeutralizationtest,NTHemagglutinationinhibitiontest,HIComplementfixation,CFELISAWesternblot第五十一頁，共六十二頁。第五十二頁，共六十二頁。ELISAforHIVantibody

MicroplateELISAforHIVantibody:colouredwellsindicatereactivity第五十三頁，共六十二頁。第五十四頁，共六十二頁。DiagnosisStrategyforViralInfection第五十五頁，共六十二頁。RapidDiagnosisforViralInfectionMorphologicalEM&IEM,occasionallyLightMicroscopy:inclusionbodyViralantigensImmunofluorescenceRadioimmunoassaysELISASpecificantibodies:IgMNucleicacids:PCR第五十六頁，共六十二頁。LaboratoryDiagnosisforFungalinfectionDirectmicroscopicCultureSerologicalNucleicacids第五十七頁，共六十二頁。MicroscopicObservationSamplesSputum,skinscrapings,nail,hair,lungbiopsy,etc.MethodTreatedwith10%KOHtodissolvetissuematerial,leav