您好,欢迎访问三七文档
当前位置:首页 > 商业/管理/HR > 咨询培训 > 微滴式数字PCR诊断检测应用方案
1EmergingApplicationsofDropletDigitalPCRinIn-vitrodiagnostics杨平|PingYangPh.D.|AssociateProductManager2Bio-Rad基因检测完整解决方案Real-TimePCRDropletDigitalPCR,ddPCRPCRAutoDGCFXConnect国械注进20163404214CFX96DeepWellDx国械注进20193220316QX200即将获得3类医疗器械注册证计划申报2类注册证3IntroductionofDigitalPCRDropletDigitalPCRWorkflowCriticalBenefitsofddPCREmergingApplicationsofDropletDigitalPCRinIn-vitrodiagnosticsNewSolutionofBio-RadinIn-vitrodiagnostics41stGeneration2ndGeneration3rdGenerationGelElectrophoresis(定性/半定量)Real-TimePCR(相对定量/绝对定量)DropletDigitalPCR(绝对定量)核酸定量技术的革新者真正的绝对定量,无需外参标准品及标准曲线,数据重复性更好更高的准确性,不再依赖PCR效率,抑制剂耐受度高,模板适用度广更高的分辨率,可识别1.1倍浓度差异,适合CNV等类型实验更高的灵敏度,适合低频突变检测、单细胞基因表达及病原微生物等一定程度上解决探针法非特异性问题以及染料法引物二聚体问题5BertVogelstein-最早的数字PCR6最早的数字PCRSchematicofDig-PCRDig-PCRofDNAfromastoolsampleNoPCRProductMutantAlleleNegativeControlWildTypePositiveControl7数字PCR时代来临DigitalPCRhitsitsstrideDigitalPCRworksbydilutingasampleintomanypartitionsandcountingupthenumberofpartitionsinwhichareactionoccurs.•Asampleisdilutedandpartitionedintohundredsorevenmillionsofseparatereactionchamberssothateachcontainsoneornocopiesofthesequenceofinterest.•Bycountingthenumberof‘positive’partitions(inwhichthesequenceisdetected)versus‘negative’partitions(inwhichitisnot),scientistscandetermineexactlyhowmanycopiesofaDNAmoleculewereintheoriginalsample.8DropletDigitalPCR•关键步骤:定量PCR体系(目的核酸片段)微分化•如何实现:PCR前油包水处理•要求:结构稳定;目的片段分布随机、独立9•阳性微滴意味着至少含有1个DNA或cDNA分子•分析软件根据每个样品中阳性微滴/总微滴的数目,基于泊松分布,计算出靶分子的浓度(copies/uL)PoissonlawofsmallnumbersSiméonDenisPoisson(1781-1840)Copiesperdroplet=微滴式数字PCR的原理10每个微滴中靶基因拷贝数的概率分布近似符合泊松分布“阳性”微液滴数(M’)出现概率:Or每一个微滴中的平均靶基因拷贝数”(λ):DNA模板数目(M)11泊松分布Sample1Sample2Sample3Sample4LowconcentrationHighconcentrationNOtargetsMediumconcentrationPoissoncorrected38/143Poissoncorrected96/143Poissoncorrected6.2/14312IntroductionofDigitalPCRDropletDigitalPCRWorkflowCriticalBenefitsofddPCREmergingApplicationsofDropletDigitalPCRinIn-vitrodiagnosticsNewSolutionofBio-RadinIn-vitrodiagnostics13DropletDigitalPCRWorkflowPartitionreagentsandsampleinto20,000dropletsPerformPCRonathermalcyclerCountdropletswithapositivePCRproduct(fluorescent)andanegativePCRproductDigitalreadoutprovidesconcentrationoftargetDNAMakeDropletsPCRDropletsReadDropletsResults“X”targetcopies14Step1—PrepareddPCRReactionMix•与现有定量PCR体系无缝对接–引物和TaqMan探针设计的原则与定量PCR相同–反应体系的准备也与定量PCR一样•专用的ddPCR预混液(2X)•FullyvalidatedPrimerPCRddPCRAssay引物、探针核酸样品ddPCR预混液15Step2—DropletsGeneration70µlOilDG8CartridgeGasketDG8CartridgeHolderDropletGenerator20µlSampleDroplets16Step3—PCRAmplification•用移液器将生成的微滴转移到PCR板相应的孔内•用铝膜封闭PCR反应板,进行扩增•扩增后微滴成为“胶囊化”的稳定状态DropletsPCR17Step4—DropletsReadingFAM/VIC/EvaGreenDropletReaderPlateHolderDropletsReading18Step5—AnalyzeResultsFAMPositiveVIC/HEXPositiveDoublePositiveDoubleNegativePositiveNegativeQuantaSoftsoftware2-Dplotview1-DplotviewEffectofpartitioningontherelativeabundanceofamutanttargetinanexcessofwild-typeDNABulkSample–20μL40,000wildtypemolecules40mutantmolecules19,960dropletsw/omutant40dropletsw/mutantPartitionedSample–20,000×1nLMutantabundance0.1%Mutantabundance33%灵敏度SensitivityQX200DropletDigitalPCRIF=51.658使用ddPCR对HIV的检测灵敏度达到LOD,3copiesper106cells4HHV6positivesin~8MhumangenomesBRAFV600EProbespecificassayschemeWTMUTFRHigh-ThroughputDropletDigitalPCRSystemforAbsoluteQuantitationofDNACopyNumber.AnalyticalChemistry.2012HighSensitiveResistanttoCrossContaminationQX200DropletDigitalPCR极端污染条件下的实测数据,验证ddPCR对抗“污染”的能力:疑问:1.微滴转移时会不会受污染?2.微滴读取时,各样本之间会不会交叉污染?验证实验设计:1.在NTC对照微滴化后人为加入不同浓度的阳性DNA模板模拟极端污染条件,是否会受影响2.将空白对照和不同浓度阳性对照于96孔板中间隔放置并交错读取微滴,是否会出现交叉干扰?结论:QX200ddPCR在微滴操作,读取分析环节不会受外界污染干扰!NTC:空白对照,水替代模板10x:某浓度的小鼠TLX基因DNA阳性对照NTC+10x:NTC微滴制备完成后,向其中加入某浓度的2μl阳性模板以模拟极端污染未受影响的NTC对照ToleranceofddPCRvsqPCRtoInhibitorySubstancesTheprobabilityofdifferencebetweenthedatasetsforddPCRandqPCRwas99.99%forbothinhibitorsandbothddPCRtargets,indicatingthatddPCRtoleratedthepresenceoftheseinhibitorsbetterthanqPCR.DingleTCetc.ClinChem.2013CNV(拷贝数变异)-精确度确定7个不同细胞系的MRGPRX1的拷贝数MRGPRX1/RNaseP(单拷贝)1.1倍梯度稀释数据实例-精确度zoomedin6MergedWells1.1-folddilutionseriesofS.aureusConstanthumangDNAPositivesPrimerdimerNegatives非特异性结合和引物二聚体问题crossreactivityspecifictarget1stGeneration2ndGeneration3rdGenerationGelElectrophoresis(定性/半定量)Real-TimePCR(相对定量/绝对定量)DropletDigitalPCR(绝对定量)核酸定量技术的革新者真正的绝对定量,无需外参标准品及标准曲线,数据重复性更好更高的准确性,不再依赖PCR效率,抑制剂耐受度高,模板适用度广更高的分辨率,可识别1.1倍浓度差异,适合CNV等类型实验更高的灵敏度,适合低频突变检测、单细胞基因表达及病原微生物等一定程度上解决探针法非特异性问题以及染料法引物二聚体问题IntroductionofDigitalPCRDropletDigitalPCRWorkflowCriticalBenefitsofddPCREmergingApplicationsofDropletDigitalPCRinIn-vitrodiagnosticsNewSolutionofBio-RadinIn-vitrodiagnostics29稀有突变检测病原微生物检测二代测序结果验证无创产前检查NIPT拷贝数变异分析液体活检应用疾病分子标志物检测数字化痕量核酸检测诊断检测应用宿主细胞DNA残留检测/质控标定/疫苗工艺开发30液体活检ResponseOriginalmutationDrugselectionpressureOriginalmutationSubclone1Subclone2•早期筛查•肿瘤基因分型•转移复发或进展的预测•疗效实时监测和评估•微小残留病检查DiagnosisDiseaseProgression•早期筛查•肿瘤基因分型•转移复发或进展的预测•疗效实时监测和评估•微小残留病检查31基因检测技术敏感度及应用32非小细胞肺癌(NSCLC)中的EGFR主要突变33肿瘤血浆游离DNA动态监测目的:采用ddPCR技术灵敏而准确地动态检测治疗阶段病人血浆中癌症相关低丰度EGFR、BRAF及KRAS基因突变(包括耐药突变)含量变化,以便调整治疗方案349个经厄洛替尼(erlotinib)一线治疗之后的NSCLC病人血浆中EGF
本文标题:微滴式数字PCR诊断检测应用方案
链接地址:https://www.777doc.com/doc-7865165 .html