数字PCR技术在肿瘤分子病理转化研究中的应用

1Theworldleaderinservingscience王俊果DigitalPCRProductManagerGeneticAnalysisDivisionLifeTechnologiesSolution数字PCR技术在分子病理临床转化研究中的应用374.219.0222WhatisDigitalPCR?（什么是数字PCR）“DivideandConquer”DigitalPCR是一种基于单分子模版PCR扩增，进行核酸拷贝数精确定量的分析方法。3dPCRWorkingPrinciple（什么是数字PCR）4dPCRWorkingPrinciple（什么是数字PCR）5微滴式数字PCR流程•将20ul含有样品的PCR预混液制备成20,000个微滴•进行emulsionPCR•PCR扩增完成后，对每个样品中的微滴逐个检测，有荧光信号的微滴为阳性微滴；反之为阴性微滴•根据记录到的阳性微滴/总微滴数目计算给出靶分子的拷贝数微滴生成油包水PCR微滴分析计算结果“X”targetcopies6ApplicationsforDigitalPCR（主要应用）GeneExpression,miRNA&CopyNumberVariationGenerationofreferencesandstandardsNGSlibraryquantificationAbsolutequantificationRarecancertargetdetectionConfirmationofNGSvariantdetectionAlleledetectionAbsolutequantificationofpathogen(e.g.viralload)DetectionofharmfulstrainsofpathogensGMOdetectionandmonitoring7vol.96no.16BertVogelstein,9236–9241,doi:10.1073/pnas.96.16.9236DevelopmentHistoryofDigitalPCR8DevelopmentHistoryofDigitalPCRPohl,G;Shih,I-M(2004).PrincipleandapplicationsofdigitalPCR.ExpertRevMolDiagn4(1):41–47.9DigitalPCRApplicationincfDNAdetectionJCONovember1,2003vol.21no.213902-3908NEnglJMed2008;359:366-377July24,2008DOI:10.1056/NEJMoa080066810Single-MoleculeDetectionofEGFRMutationsinPlasmaClinCancerRes2009;15(6)March15,200911AnnalsofOncology25:2304–2313,2014DigitalPCRApplicationincfDNAdetection12UseDigitalPCRinQuantifyT790MMutationCANCERGENOMICS&PROTEOMICS1:31-38(2015)Ahypothesisregardingacquiredtyrosinekinaseinhibitor(TKI)resistanceusingdigitalPCR13IdentificationofEGFRT790Minapatientplasmasample=catprod14DetectionOfRareSomaticMutationDownTo0.01%•Dr.AtochaRomerofromHospitalClinicodeMadrid•LookedatPIK3CAH1047RmutationincfDNAA)Samplewithmutation(positive)B)Normalsample–nomutationpresent“…andwehaveobservedthatthesensitivitywecanachievewiththisassayisfarsuperiorthanwithotherapproaches.Thisassaysmeetsourrequirementsforquantifyinglowfrequencymutationsincirculating-freeDNA.”16AccurateQuantificationofGeneExpressionChanges0 10000 20000 30000 40000 50000 60000 T0 15 min exposure 15 min exposure, 1hr withdrawal 15 min exposure, 6hr withdrawal Transcript copies/ul pri-mir9-1 pri-mir9-1 pri-mir9-2 pri-mir9-2 pri-mir9-3 pri-mir9-3 0 10000 20000 30000 40000 50000 60000 T0 6 hr exposure 6 hr exposure, 6 hr withdrawal 6 hr exposure, 24hr withdrawal Transcript copies/ul pri-mir9-1 pri-mir9-1 pri-mir9-2 pri-mir9-2 pri-mir9-3 pri-mir9-3 dPCRonQuantStudio™3Ddetectedclearandreproduciblechangesinabundanceofsequenceproducedbyonlyoneofthethepri-miRNAlociReplicatemeasurementswerealmostidentical.Differencesinexpressionlevelsofrelatedsequenceswerereadilydetectable.DatacourtesyofDr.AndrePietrzykowski,RutgersUniversity