应用DHPLC技术进行诊断性分析的质量保证体系培训课件.ppt

应用应用DHPLCDHPLC技术进行技术进行诊断性分析的质量保诊断性分析的质量保证体系证体系诊断性分析的要求诊断性分析的要求n临床分子遗传学分析的复杂性临床分子遗传学分析的复杂性n临床分子检测结果的一致性和精确性临床分子检测结果的一致性和精确性n变性高效液相色谱变性高效液相色谱(DHPLC)DHPLC)作为一种高效和敏作为一种高效和敏感的基因突变检测技术感的基因突变检测技术nDHPLCDHPLC技术质量控制技术质量控制2应用DHPLC技术进行诊断性分析的质量保证体系AMERICAN COLLEGE OF MEDICAL GENETICSStandards and Guidelines for Clinical Genetics Standards and Guidelines for Clinical Genetics LaboratoriesLaboratories2005 EditionG:CLINICAL MOLECULAR GENETICSG:CLINICAL MOLECULAR GENETICS These Standards and Guidelines specifically refer to the use of molecular techniques to examine heritable or somatic changes in the human genome.G18G18Denaturing High Performance Liquid Chromatography(dHPLC)Denaturing High Performance Liquid Chromatography(dHPLC)(Section Added November 2003)(Section Added November 2003)3应用DHPLC技术进行诊断性分析的质量保证体系CMGSBestPracticeGuidelinesUseoftheWAVESysteminDiagnosticServicePreparedandeditedbyJohnHarvey,NationalGeneticsReferenceLaboratory(Wessex),Salisbury,UKandElsSchollen,CentreforHumanGenetics,Leuven,Belgiumlastupdate:12March2004IntroductionLaboratoryprocessDHPLCsystemDataqualityChecking&reportingguidelinesReferences4应用DHPLC技术进行诊断性分析的质量保证体系5应用DHPLC技术进行诊断性分析的质量保证体系DHPLC SOPsnInstrumentormaintenanceSOPuTechniquenGeneralDHPLCSOPWAVE3500,3500HTuMethodnDisease-specificSOPsRett,BRCA,HNPCCMarfan,uApplicationcompany+usersgeneral users+companyspecific users6应用DHPLC技术进行诊断性分析的质量保证体系SupplementaryAppendix1STANDARDOPERATINGPROCEDUREWAVESystemOperationandMaintenanceSOP-O&MWAVESystemOperationandMaintenanceForWAVESystemModels3500,3500Aand3500HT7应用DHPLC技术进行诊断性分析的质量保证体系WAVESystemOperationandMaintenance AnalysisoftheWAVELow&HighRangeMutationStandardsThemaintenanceprocedureDNASepandDNASepHTcartridgemaintenance8应用DHPLC技术进行诊断性分析的质量保证体系RoleofMutationstandards:checkingofcorrectfunctioningoftheWAVESystem,includingovencalibration,cartridgeperformance,buffercompositionandstability,toensurereproducibilityandaccuracyofthechromatographicanalysis.Mutationstandardsberunwhen:lTheroutinepre-run,lWeeklyandmonthlymaintenanceprocedure,lAfterreplacementofanycomponent,lValidationforanewbatch,lAsanassaycontrol,atthebeginningandendofeveryrun,preferablyalsoafterevery100injectionsforlongruns.AnalysisoftheWAVELow&HighAnalysisoftheWAVELow&HighRangeMutationStandardsRangeMutationStandards 9应用DHPLC技术进行诊断性分析的质量保证体系10应用DHPLC技术进行诊断性分析的质量保证体系Normalrangesofthemutationstandards11应用DHPLC技术进行诊断性分析的质量保证体系The maintenance procedure3.1Filterandflush3.2Pre-runmaintenance3.3Weeklymaintenance3.4Quarterlymaintenance3.5Othermaintenanceoperations3.6Preventativemaintenanceprocedureandsystemvalidation12应用DHPLC技术进行诊断性分析的质量保证体系Filterandflush nTheprincipleoffiltrationinvolvespreventingunwantedcontaminantsfromenteringthesystem.nFiltrationappliestotwospecificareas:solventfiltrationandin-linefiltration.nThesystemflushingistoremovemobilephasesaltcomponentsthatcanprecipitateunderstrongsolventconditions.13应用DHPLC技术进行诊断性分析的质量保证体系Pre-run maintenance1.Buffercheck2.Injectionsystemwashing3.Pressurecheck4.Checktheabsorbanceonthedetector5.Purgethelines14应用DHPLC技术进行诊断性分析的质量保证体系Weekly maintenancenInlinefilterreplacementnCheckthesyringe15应用DHPLC技术进行诊断性分析的质量保证体系Quarterlymaintenance nCheckUVlampnUVlampreplacementnCleaningthesystem(Isopropanolcleaning)16应用DHPLC技术进行诊断性分析的质量保证体系DNASepandDNASepHTcartridgemaintenance 1.RegularmaintenancescheduleEvery96-192injections:ExtendedhotwashEvery1000injections:ReversehotwashDNASepwash(ifreversehotwashfailstoresolvemutationstandards)2.Short-termcartridgestorage3.Long-Termcartridgestorage4.Newcartridgeinstallation17应用DHPLC技术进行诊断性分析的质量保证体系Daily MaintenancenEquilibratethecartridge50%A50%Bfor15minutesRun1-2blanksnVerifysystemperformance(pre-analysis)RunastandardnRunSamplesnVerifysystemperformance(postanalysis)18应用DHPLC技术进行诊断性分析的质量保证体系Weekly MaintenanceAnExtendedActiveCleanWashisrecommendedevery100injections.(Usuallydoneaftereach96wellplate)OvenOvenSetto:Setto:80C80CPumpPumpSetto:Setto:100%D100%D15-3015-30minutesminutesWASHWASHOvenOvenSetto:Setto:56C56CPumpPumpSetto:Setto:50%A-50%B50%A-50%BEQUILIBRATEEQUILIBRATE45-9045-90minutesminutesRunStandardstoVerifySystemPerformance!RunStandardstoVerifySystemPerformance!19应用DHPLC技术进行诊断性分析的质量保证体系1000 Injection Maintenance AReverseHotWashisrecommendedevery1000injectionsUV/FLDetector TurnofftheTurnoffthepumppumpReversetheReversethecartridgecartridgedirection.direction.SettheovenSettheovento80C.to80C.SetthepumpSetthepumpto100%D.to100%D.60-9060-90minutesminutes20应用DHPLC技术进行诊断性分析的质量保证体系Storing the CartridgenFlushthecartridgewith100%DBuffer.nRemovethecartridgefromtheWAVESystem.nCapthecartridgewithendplugs.nStorethecartridgeatroomtemperature.21应用DHPLC技术进行诊断性分析的质量保证体系Installing a New Cartridge1.1.StopthepumpflowStopthepumpflowandremovetheoldandremovetheoldcartridge.cartridge.2.2.RemovetheplugsRemovetheplugsfromthenewfromthenewcartridge.cartridge.3.3.InstallthenewInstallthenewcartridgewiththecartridgewiththearrowpointingarrowpointingtowardtherearoftowardtherearoftheoven.theoven.UV/FLDetector22应用DHPLC技术进行诊断性分析的质量保证体系1.1.Makesuretheovenheatsuptoatleast40C.Setthepumpto100%DMakesuretheovenheatsuptoatleast40C.Setthepumpto100%D0.500mL/min.0.500mL/min.2.2.Ensurethepressureisstableandgraduallyincreasetheflow(0.9mL/minorEnsurethepressureisstableandgraduallyincreasetheflow(0.9mL/minor1.5mL/min).1.5mL/min).3.3.Flushthecartridgefor15minutes.Flushthecartridgefor15minutes.4.4.Setthepumpto50%BufferA,50%BufferBandequilibratefor30minutes.Setthepumpto50%BufferA,50%BufferBandequilibratefor30minutes.Equilibrating a New Cartridge100%100%50%50%50%50%23应用DHPLC技术进行诊断性分析的质量保证体系Verify New Cartridge PerformanceLow-RangeMutationLow-RangeMutationStandardStandardDNASizingControlDNASizingControlStandardStandard24应用DHPLC技术进行诊断性分析的质量保证体系SupplementaryAppendix2STANDARDOPERATINGPROCEDUREDHPLCSOP-DHPLCDHPLCmutationdetectiononTransgenomicWAVESystem3500Wavemaker4.1.44&HSM3.0-2.1(build2)Navigator1.5.4(build19)25应用DHPLC技术进行诊断性分析的质量保证体系PCRrequirementsnPrimerdesignnTemplatepurityandconcentrationnDNAPolymerasesnPCRbuffermixnPCRplatesnPCRqualityandProductmixingnPost-PCR,filmusenControls26应用DHPLC技术进行诊断性分析的质量保证体系Primer DesignnUseaprimer-pickingprogramnPrimersshouldideallybenocloserthan30-50bpfromtheendofthesequencetobeanalyzedformutationsnPrimersshouldbe18-30bpinlengthnTheTmdifferencebetweenprimersinapairshouldideallybelessthan2C.TransgenomicWAVESystemCustomerscanusetheTransgenomicWAVESystemCustomerscanusetheAdvancedFeaturesofAmpliconDesignatAdvancedFeaturesofAmpliconD27应用DHPLC技术进行诊断性分析的质量保证体系Size of PCR fragment nTheoptimalsizerangefordetectingmutation/SNPsbyDHPLCwith100%accuracyis150-500bp.nFragments500bpcanbegeneratedbutsensitivitydecreasedandtimeofelutionincreased.nForfragments150bp,differenceofmeltingpointbetweenfragmentstoonarrow(thefragmentsmeltovertoonarrowatemperaturerange).28应用DHPLC技术进行诊断性分析的质量保证体系Quantity of PCR fragment nThePCRproductshouldbesufficientlyconcentratedthat2lrunonanagarosegelproducesaclearlyvisibleband(20ng/l)nDilutesamples(verylowyields)producepoorqualityresults(poorsignal:noiseratio).nVeryhighyieldscanleadtolargeproportionsofmisincorporationsandhenceincreaseddifficultyincallingmutations.nUsually3-10l(50-200ng)ofunpurifiedPCRproductwouldbeinjectedontothecolumn(peronetemperature).nA8lminimumaliquotofPCRproductshouldbesuppliedinPCRtubesinstripsof8(peronetemperature).29应用DHPLC技术进行诊断性分析的质量保证体系Sample Preparation for DHPLCnDNAmustbeclean,allcellulardebrisandorganiccompoundsmustberemoved.nSaltingoutmethodispreferred.nDNAextractedwithsomecommercialsystemsmaybedilutedto10ngtoreduceeffectsofimpuritiestoPCR.nDNAoflowqualitywillresultinsub-optimalPCRresults(henceDHPLCprofiles).DNAquality&concentrarion30应用DHPLC技术进行诊断性分析的质量保证体系Table1.RecommendedcleaningproceduresforDNAextraction.IsolationMethodRecommendedAdditionalCleaning:OrganicExtraction(e.g.phenol/chloroform)Chloroform/isoamylback-extractionfollowedbyethanolprecipitationandwashChaotropicSalts(e.g.guanidiniumisothiocyanate)EthanolprecipitationandwashSpinColumnEthanolprecipitationandwashTable2.RecommendedDNAquantitiesusedforPCR(50Lreaction).TemplateRecommendedQuantityHumangenomicDNA50-200ngPhageDNA1-10pgPlasmidDNA0.1-1.0pg31应用DHPLC技术进行诊断性分析的质量保证体系The Importance of Polymerase Fidelity for Mutation DetectionnImportanceofhighfidelityindHPLC500bpWildTypeFragmentRedTraceOptimasePolymeraseGreenTrace9:1MixAmplitaqGoldandPfuTurboHeteroduplexduetomisincorporation32应用DHPLC技术进行诊断性分析的质量保证体系Polymerase Fidelity Comparison33应用DHPLC技术进行诊断性分析的质量保证体系Maximum recommended concentrationsof acceptable PCR additivesAcceptableadditives(maximumfinalconcentration)Additiveswherefinalconcentrationmustbe2mVatA260.nPeaksofintensity30%oftheaveragepeakintensity.nWeakpeaksaremorelikelytoleadtofalse-negative/positiveresults.Minimum peak intensity45应用DHPLC技术进行诊断性分析的质量保证体系Identification of sequence variantsnThepresenceofheteroduplexesisoftendetectedasachangeinthenumberofpeaks(maybe2,3or4peakpattern).nTwopeakpatternsaccountforthemajorityofmutations.nCompleteresolutionofthe2heteroduplexesisnotalwaysnecessary.nMutationsmayappearonlyasaslightbroadeningofthesinglepeak,orasasubtlechangetoashoulderonthepeak.nAllsamplesidentifiedasheteroduplexesbyDHPLCanalysismustbesequencedinbothdirectionstoconfirmanddeterminethenatureofthesequencechange.46应用DHPLC技术进行诊断性分析的质量保证体系nThehomoduplexwild-typepatternistypically1peak,butmaybe2peaks,dependinguponthemeltingprofile.nElutionprofilesthatdifferfromthewild-typeindicatethepresenceofDNAsequencechanges.Butthemutationtypecannotbepredictedfromtheheteroduplexpattern.nEachmutationinagivenPCRfragmentispredictedtohaveauniqueheteroduplexpattern(highlyspecificelutionprofile).Thisisusefulforquickgenotypingofunknownsamplesbycomparisonwithpositivecontrolsamples.nHowever,traceprofilesarenotalwaysuniqueforaspecificmutation,i.e.differentDNAvariantscangiveidenticalprofiles.nChangesinretentiontimedonotaccuratelypredictthepresenceofasequencechange.Trace specificity47应用DHPLC技术进行诊断性分析的质量保证体系Datachecking,reportingandstorage nDatacheckingnPositiveresultsnFalsepositiveresultsnNegativeresultsnFalsenegativeresultsnSensitivitynDetectionofmosaicsnArchiving48应用DHPLC技术进行诊断性分析的质量保证体系SupplementaryAppendix3STANDARDOPERATINGPROCEDUREMECP2SOP-MECP2DHPLCscreeningofMECP2InthecontextofRettSyndrome49应用DHPLC技术进行诊断性分析的质量保证体系Materials nWorksheet:n-LotNo.ofallproductsn-Equipmentidentifiersn-Patient-identifiern-Performingtechnician(s)n-Dateofexperiments51应用DHPLC技术进行诊断性分析的质量保证体系MaterialsnPCR-StandardPCRequipment(Location xxx)-OptimaseDNApolymerase(2.5U/l)(Location xxx)-OptimasePCRbufferwithMg2+(Location xxx)-Primers(Eurogentec)(stock)as250pmol/l.(appendixA)(Location xxx)-Primerworksolutionscontain2.5pmol/lofeachprimer(Location xxx)-PuredNTPs(withoutdUTP)(2mM)(Location xxx)-PCRsystem52应用DHPLC技术进行诊断性分析的质量保证体系Materials nDHPLCsystem-StandardDHPLCmaterial(forpartnumbersseeappendixCinSOP-O&M)-WAVESystem3500HT,WAVEMAKER4.1.44&HSM3.0-2.1build2.nPatientmaterial-Patient DNA-Positive controls-Negative controls-Normal controls53应用DHPLC技术进行诊断性分析的质量保证体系UsesofPlasmidControlsasReferenceReagentsnoethicalproblemsrenewableresourceCanusesamereferencereagentascontrolforPCR,heteroduplexandmutationdetectionanalysisUniversalreagentswhichcanbeincorporatedinQCproceduresandSOPsAdvantages:ValidationofnewprotocolsExonspecificwildtypeandmutatedcontrolsforexistingassaysValidationoftransferofprotocolsbetweenmachines/labsUses:54应用DHPLC技术进行诊断性分析的质量保证体系Method nPre-PCRPCRCompositionPCRConditionsnPostPCRHeteroduplexformationAgarosegelelectrophoresisnDHPLC55应用DHPLC技术进行诊断性分析的质量保证体系Interpretationoftheresults nThemutationstandardsatthebeginningandendoftherunareevaluatednAllpositivecontrolsshouldbevisibleattheirspecifictemperaturenIfoneofthecontrolsdoesnotfulfillthecriteria,negativeresultsarenotvalidandhavetoberepeated.Positiveresultscanbeprocessedasusual.nTheelutionprofilesofaspecificfragmentfromthedifferentpatientsarecomparedwitheachotherandscoredaccordingtothegeneralDHPLCcriteria.Theminimumpeakheightmustbe2mV.Anyparticularobservationshouldbenotedonworksheetsortechnicalreports.nAmpliconswithanaberrantelutionpatternarere-analysedbydirectsequencingonanindependentamplicon56应用DHPLC技术进行诊断性分析的质量保证体系Interpretationoftheresults nAllprematuretruncationmutationsareimmediatelyreportable.nThepathogenicityofthemissensemutationswilldependonthepositionandthetype.Interpretationisthensubjecttogoodpracticeandliteraturereview.MutationsinthetwohighlyconservedMeCP2domains,themethylbindingdomainandthetranscriptionrepressiondomain,arelikelytobecausative.nIfamutationisofunknownsignificance,samplesshouldbeobtainedfromthepatientsparents.Ifthemutationisfoundtobede novo,itislikelytobecausative.Ifthemutationispresentinthemother,X-inactivationstudiesneedtobecarriedoutonthemotherofthepatientIfboththemotherandthedaughterhaverandomX-inactivationthemutationisunlikelytobecausative.57应用DHPLC技术进行诊断性分析的质量保证体系Reportingprocedures nNEGATIVERESULTINAFEMALEnNEGATIVERESULTINAMALEnNORMALPARENTnPOSITIVERESULTINAFEMALE58应用DHPLC技术进行诊断性分析的质量保证体系NEGATIVERESULTINAFEMALE Rett syndrome is caused by mutations in theMECP2gene.Molecularanalysisofthisgenehasbeen carried out on patient*,however nocausativemutationhasbeenfound.DHPLCanalysiswasusedtoscreenformutationsintheMECP2gene.Thistechniquehasasensitivityof95%forthedetectionofpointmutations,microdeletionsandmicroinsertionsbutwillnotdetectgrossdeletionsofentireexons.Only80%ofRettsyndromepatientshaveadetectablemutationwithintheMECP2gene.59应用DHPLC技术进行诊断性分析的质量保证体系NEGATIVERESULTINAMALE Molecular analysis of the MECP2 gene has beencarriedoutonpatient*.Howevernocausativemutationhasbeenfound.DHPLCanalysiswasusedtoscreenformutationsintheMECP2gene.Thistechniquehasasensitivityof95%forthedetectionofpointmutations,microdeletionsandmicroinsertionsbutwillnotdetectgrossdeletionsofentireexons.ThefrequencyofMECP2mutationsinmentallyretardedmalesisestimatedat0.2%60应用DHPLC技术进行诊断性分析的质量保证体系NORMALPARENT*istheparentof*,apatientwithRettsyndromewhohasthe*mutationintheMECP2gene.TheparentsDNAhasbeenanalysedforthisspecificmutationbyThereisnoevidenceofthe*mutationintheperipheralbloodsampleof*.However,wecannotruleoutgermlinemosaicismforthismutation.Prenataltestingforthismutationisapossibility.61应用DHPLC技术进行诊断性分析的质量保证体系POSITIVERESULTINAFEMALE*hasbeenfoundtocarry*atbase*oftheMECP2gene(*).ThisresultconfirmsthediagnosisofRettsyndromein*.Morethan95%ofRettsyndromecasesaresporadic,however*smothermaybeacarrierofthismutation.Also,oneoftheparentsmayhavegermlinemosaicismforthismutation.62应用DHPLC技术进行诊断性分析的质量保证体系诊断性诊断性DHPLCDHPLC质量保证（质量保证（DDQADDQA）nWAVEWAVE系统的操作和维护系统的操作和维护SOPSOP；nDHPLCDHPLC技术分析基因突变的通用技术分析基因突变的通用SOPSOP；nDHPLCDHPLC检测特定疾病基因的检测特定疾病基因的SOPSOP：RettRett综合征综合征相关的相关的MECP2MECP2基因筛查中基因筛查中SOPsSOPs。63应用DHPLC技术进行诊断性分析的质量保证体系DHPLC技术性能发挥技术性能发挥1.1.首先，只有经过充分的实验设计，条件优化，和实首先，只有经过充分的实验设计，条件优化，和实际验证后才能达到这种高灵敏度；际验证后才能达到这种高灵敏度；2.2.第二，严格的质量控制，持续的系统验证，和严格第二，严格的质量控制，持续的系统验证，和严格的实验程序也是达到这种高灵敏度必不可少的；的实验程序也是达到这种高灵敏度必不可少的；3.3.第三，操作者的经验也不可低估；第三，操作者的经验也不可低估；4.4.第四，即便在最理想的条件下，也不能保证对所有第四，即便在最理想的条件下，也不能保证对所有的扩增产物达到的扩增产物达到100%100%的灵敏度；的灵敏度；5.5.最后，虽然不是直接由该方法引起，但必需意识到最后，虽然不是直接由该方法引起，但必需意识到DHPLCDHPLC分析的是小片段的分析的是小片段的PCRPCR产物，因而只能检测单产物，因而只能检测单碱基的突变，小片段的插入和缺失，大片段的插入碱基的突变，小片段的插入和缺失，大片段的插入和缺失不能检测。和缺失不能检测。64应用DHP