ddPCR原理与应用简介

DropletdigitalPCR(ddPCR)微滴式数字PCR原理与应用简介工作原理技术优势医学应用工作原理Part1PCR发展历程•普通PCR•定性分析第一代•Real-timePCR•间接定量分析•依赖Ct或Cq值，标准品第二代•数字PCR•直接定量分析•无需标准品•绝对定量第三代将反应体系加入微滴发生卡中目的片段和背景序列随机分布到微滴内将微滴化处理的样品转移至96孔PCR反应板中，并封膜，进行PCR扩增反应体系配制微滴制作微滴PCR扩增读取分析结果将反应板置于微滴分析仪内，对微滴荧光信号逐个检测、分析样品、引物和探针/EvaGreen染料与预混液混合ddPCR的工作流程：QX200ddPCRddPCR的工作流程：QX200ddPCRddPCR的工作流程：QX200ddPCRddPCR的工作流程：QX200ddPCRTaqMan探针EvaGreen染料ddPCR的工作流程：QX200ddPCRddPCR的工作流程：QX200ddPCR技术优势Part2ddPCR技术优势实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%准确度、精密度和重复性较高，可精确测定靶基因的相对表达、CNV分析等终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制剂的影响，因而对样本的复杂性容忍度较高01020304无需标准品，即可对动样品中的DNA进行绝对定量微滴化实现了稀有序列或稀有突变碱基的富集ddPCR技术优势实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%准确度、精密度和重复性较高，可精确测定靶基因的相对表达、CNV分析等01020304无需标准品，即可对动样品中的DNA进行绝对定量终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制剂的影响，因而对样本的复杂性容忍度较高Purpose&ExperimentalDesign：Inthisstudy,weconstructedaplasmidcontainingtheamoAgeneofAOA(A-amoA)andAOB(B-amoA),andthenosZandnirSgenesofdenitrifiers.DropletdigitalPCR(ddPCR)wasevaluatedforcharacterizationoftheplasmidreferencematerial.Results:ThecopynumberconcentrationofthedigestedplasmiddeterminedbyddPCRagreedwellwiththatdeterminedbyLC–IDMS,improvingboththeaccuracyandreliabilityoftheplasmidreferencematerial.Conclusion:ThismethodhasthepotentialtoaccuratelyquantifyDNAreferencematerials.ddPCR技术优势实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%准确度、精密度和重复性较高，可精确测定靶基因的相对表达、CNV分析等终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制剂的影响，因而对样本的复杂性容忍度较高01020304无需标准品，即可对样品中的DNA进行绝对定量Purpose&ExperimentalDesign：Wedescribeaseriesofexperimentstocomparetheinhibitiontoleranceoflaboratory-developedCMVqPCRandddPCR(Bio-RadLaboratories,Hercules,CA,QX-100)assaysbyintroducingapanelofclinicallyrelevantinhibitors(SDS,EDTAandheparin)directlyintothePCRreactionsConclusion:ddPCRmayofferanadvantageoverqPCRwhendealingwithinhibitionpronespecimens.ddPCRmayproveespeciallyusefulforclinicalsampletypessuchasstool,sputum,andtissueareknowntobemorerecalcitranttoremovalofinhibitorsthroughtypicalextractionmethods.ddPCR技术优势实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%准确度、精密度和重复性较高，可精确测定靶基因的相对表达、CNV分析等01020304无需标准品，即可对样品中的DNA进行绝对定量终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制剂的影响，因而对样本的复杂性容忍度较高Results:OurcomparisonofmicroRNAquantificationbyddPCRandreal-timePCRrevealedgreaterprecision(coefficientsofvariationdecreasedby37-86%)andimprovedday-to-dayreproducibility(byafactorofseven)ofddPCR.Conclusion:Nanoliter-sizeddroplettechnologypairedwithdigitalPCR(ddPCR)holdspromiseforhighlyprecise,absolutenucleicacidquantification.Results:Theresultswereconsistentwiththeresultspresentedabove,ddPCRshowedgreaterprecision.医学应用Part3Purpose:TodeterminewhetherddPCRcouldidentifythedevelopmentofresistancemutationsaftertreatmentwithtargetedtherapy,westudiedpatientswithadvancedEGFR-mutantNSCLCtreatedonaprospectiveclinicaltrialoffirst-lineerlotinib(NCT00997334).稀有突变碱基或稀有序列的检测Results:All9patientsexhibitedaplasmaresponsetoerlotinib,with8demonstratingacompleteplasmaresponse.In6ofthepatients,plasmalevelsofmutantEGFRwereagaindetectedatobjectiveprogression,withplasmaprogressiondetected4-12weekspriortoRECISTprogression.Theremaining3patientshadnoreemergenceofplasmagenotypeatobjectiveprogression.Conclusion:DropletdigitalPCRisanattractivetechnologyasitsspeed,cost,andeaseofuseissimilartootherPCR-basedassays,yetthesensitivityandquantitativenatureofthisassayoffersbroaderclinicalapplication.稀有突变碱基或稀有序列的检测拷贝数变异检测Purpose:ComparebiopsyDNAtestedwithFISHtoplasmaDNAtestedwithdropletdigitalPCRtoidentifyknownHER2amplifiedandnon-amplifiedsamples.Conclusion:digitalPCRofplasmaDNAhashighaccuracyinthedeterminationofHER2status.Thisapproachcouldbeadaptedtotheassessmentofanyamplifiedlocusincancerandinparticularmaybeausefulstrategyscreeningforpotentiallyrareacquisitioneventsinresponsetotherapy.Results:ddPCRwasusedtodetectHER2copynumberamplificationsinbothcell-linesandcirculatingtumorDNAfromplasma.AllsampleshadpreviouslybeentestedusingFISH.ddPCR与NGSPurpose:Wehavedevelopedanewassay“QuantiSize”capableofconcurrentlymeasuringtheabsoluteconcentrationandthelengthofunknownamplifiableDNAtemplates,makingitwellsuited.Conclusion:ThefrequencydistributionshowsahighdegreeofoverlapandacommoncenterpointfortheestimationmadeusingtheQuantiSizeassayandtheobservationsfromtheMiSeq.TheQuantiSizeassayhasthepotentialtoincreasetheaverageyieldofusabledatageneratedfromsequencingruns,therebyincreasingtheefficiencyandthroughput.Experimentaldesign:Thisassayexploitsacorrelation,reportedherein,betweenthelengthofanamplifiedDNAmoleculeandthefluorescenceamplitudeproducedindropletdigitalPCR(ddPCR),toallowtheusertocalculatethesizeofunknownDNA.ddPCR与NGSThanksforYourAttention!AnyQuestions?