法庭科學(xué)SWGDAM驗證流程課件

法庭科學(xué)SWGDAM驗證流程

議程SWGDAM介紹華夏TM白金SWGDAM驗證YfilerPlusTMSWGDAM驗證ISO18385介紹SWGDAM主頁Source:關(guān)于SWGDAMFirstmeetingofTechnicalWorkingGrouponDNAAnalysisMethods(TWGDAM)washeldinNovember1988,withasubcommitteeonrestrictionfragmentlengthpolymorphism(RFLP)DNAanalysis.TWGDAMcontinuedtoprovidealevelofdirectiontotheforensicDNAcommunitybyissuingguidelinesforDNAanalysis,andtheguidelinesbecamedefactostandardsandwererecognizedbycourts.SWGDAMChairmanisappointedbytheFBIDirector,andmembersareselectedbytheChairmanfromforensicDNAlaboratories.SWGDAMhasmultiplecommitteesincludingAutosomalSTRCommittee,CODISCommittee,RapidDNACommittee,NextGenerationSequencingWorkingGroup,etc.OneofSWGDAM’smostimportantresponsibilitiesistherecommendationofrevisionstotheFBI’sQualityAssuranceStandards(QAS)forDNAAnalysis.SWGDAM出版物SWGDAM法醫(yī)DNA分析方法驗證指導(dǎo)3.7PopulationStudies3.8MixtureStudies3.9PCRBasedStudies3.3Sensitivity3.4Stability3.5.Precision&Accuracy3.6CaseTypeSamples3.2SpeciesSpecificity3.1GeneticMarkersSWGDAMValidationGuidelinesforForensicDNAAnalysisMethodsapprovedinNovember2012華夏白金物種特異性–非靈長類10ngnon-primateDNA,or105copiesofmicrobes(speciescommonlyfoundintheoralcavity,listedinUserGuide)wereaddedtothesystemusing29PCRcycles.Lowpeaksweredetectedwithbovine,dog(likecontamination,alsofoundintherepeat),horse,mouseandpig.BovineChickenDogHorseMicrobesMousePigRabbitRatSheepNTC200rfu200rfu700rfuSWGDAM3.2Theabilitytodetectgeneticinformationfromnon-targetedspecies(e.g.,detectionofmicrobialDNAinahumanassay)shouldbedetermined.華夏白金一次性成功通過率Allsampletypeshave≥90%passrate(PAT=175rfu)with1Proportiontest.TherecommendedrxnvolumeforHXPis25ul,sometestshereweredonewith10ulreactionvolumetochallengethesystem.SWGDAM3.4TheabilitytoobtainresultsfromDNArecoveredfrombiologicalsamplesdepositedonvarioussubstratesandsubjectedtovariousenvironmentalandchemicalinsultsshouldbeevaluated.樣本準(zhǔn)在3500xl上的準(zhǔn)確度Sizedeviationof42populationsamplesshoweddeviation<0.50bpforallallelesSWGDAM3.5Precisionandaccuracyoftheassayshouldbedemonstrated.Accuracyisthedegreeofconformityofameasuredquantitytoitsactual(true)value.遺傳標(biāo)記在不同種族的人群中的分布

SWGDAM3.7Thedistributionofgeneticmarkersinpopulationsshouldbedeterminedinrelevantpopulationgroups.AllelefrequencyformajorChinesepopulationgroupswasstudied.DataofD6S1043waspresentedabove,andthedatawithothermarkerswaspublishedinUG.反應(yīng)預(yù)混物保護(hù)帶研究Eightrawmaterialsinmastermixwerevariedupto20%.ICBwasabove35%formostrawmaterialswith10%oreven20%variation.Detaildataincludingcapabilityanalysispresentednext.ComponentconcentrationsIndividualcomponentsTheHuaxiaPlatinumMMisrobusttorawmaterialchangeSWGDAM3.9Thereactionconditionsneededtoprovidetherequireddegreeofspecificityandrobustnessshouldbedetermined.SWGDAM對Y-STR的指導(dǎo)Y-STRtypingisalsousedinlieuofautosomaltypingforthedetectionofmaleDNAinmixturesthatcontainanoverabundanceoffemaleDNA.Giventhatundercertainconditionsamaleminorcontributorinamixtureoffemale:maleDNAmayonlybedetectablebyY-STRtyping,laboratoriesshouldpursueY-STRanalysisasthemostappropriatemeansofdetectingamalecontributor(s)insomeforensicsamplesInterpretationGuidelinesforY-ChromosomeSTRTypingapprovedinJanuary2014ThisSWGDAMstatementhighlightstheimportanceofmalespecificityofY-STRkitwiththemalefemalemixturescommonlyfoundwiththesexassaultcases.PrimermixDesign/screenprimerpairswithminimalfemalecross-reactivityMastermixLowertheMgandTaqenzymeconcentrationstominimizefemalecross-reactivityMultipleroundsofDOEwereperformedtooptimizetheMMcomponentsPCRconditionIncreasetheannealing/extensiontemperaturetoincreasemalespecificity提高Y-特異性的工作YfilerPlus在男女混合DNA的表現(xiàn)YfilerPlusrecoveredfullprofilewith1:1000male:femalemixtureNoextrapeakswithYfilerPlus,whilemultipleextrapeakswithPPY23andYfilerInternalProductSpec:Thekitwillrecoverfullprofilewithaverage40%ICBwhenamplifiedwithmale:femalemixturesof1:1000YfilerPlus高濃度女性DNA下的基線200rfuLowfemalecross-reactivity(<175PAT)withinthereadregionarealldocumentedintheUG.Blue271Red139/144RedgrassOutsidethereadregion68bp406bpInternalProductSpec:TheYfilerPluskitshallnotamplifyreproduciblefemaleDNAinthe1ugrange,withoutreproducibledropins&artifactswithinthereadregion.3微克女性DNA在三個盒子的比較3ugofDNAfrom10differentfemaledonorsweretestedwiththeYfilerPlus,PPY23andYfilerkits.Thehighestreproduciblefemaleartifactsfromeachsamplewereplottedbykit.YfilerPlusandYfilerhavesignificantlylowerartifactpeakheightsthanPPY23.PAT新的法醫(yī)DNA標(biāo)準(zhǔn)–ISO1838