RPA——PCR技术的革命.ppt

RPA PCR技术的革命 什么是RPA 为什么说RPA是一场技术革命 应用及相关 什么是RPA 自PCR技术诞生以来已经有三十年了 从经典PCR 实时定量PCR再到现在的数字PCR 这一技术在不断蜕变却从未淡出我们的视野 RPA RecombinasePolymeraseAmplification 全称重组酶聚合酶扩增技术 被称为可以替代PCR的核酸检测技术 RPA技术主要依赖于三种酶 能结合单链核酸 寡核苷酸引物 的重组酶单链DNA结合蛋白 SSB 链置换DNA聚合酶 这三种酶的混合物在常温下也有活性 最佳反应温度在37 C左右 RPA的原理 PrevioulyestablishedRPAConditions 2006 引物设计RPA分析的关键在于扩增引物和探针的设计 PCR引物多半是不适用的 因为RPA引物比一般PCR引物长 通常需要达到30 38个碱基 引物过短会降低重组率 影响扩增速度和检测灵敏度 在设计RPA引物时 变性温度不再是影响扩增引物的关键因素 RPA的引物和探针设计不像传统PCR那样成熟 用户需要自己摸条件进行优化 Speed10to15minutedetectiontimeSensitivitySinglemoleculeCostCheapaccessaslittleornohardwareisrequiredSimplicityStabilisedreactionformat minimalsamplepreparationRobustnessTosamplecontaminantsandtemperaturefluctuationsPortabilityHandheldinstrumentationordisposabletestformat 为什么说RPA是一场技术革命 RPA具体应用举例 Clinical Foodsafety Agricultural Bloodbankscreening Environmental Animalhealth RecombinasePolymeraseAmplification RPA ofCaMV 35SPromoterandnosTerminatorforRapidDetectionofGeneticallyModifiedCrops ChaoXu1 LiangLi1 2 WujunJin1 2andYusongWan1 2 1BiotechnologyResearchInstitute ChineseAcademyofAgriculturalSciences Beijing100081 China E Mails xuchao1667 C X liliang L L jinwujun W J 2InspectionandTestingCenterforEnvironmentalRiskAssessmentofGeneticModifiedPlant RelatedMicroorganisms Beijing MinistryofAgriculture Beijing100081 China 1 Introduction TheInternationalServicefortheAcquisitionofAgri biotechApplications ISAAA estimatesthatmillionsoffarmerscultivatedgeneticallymodified GM cropsovermorethan170millionhectaresacross27countriesin2013 themajorGMcropspecieswerecanola maize cotton andsoybean 1 Althoughthepolymerasechainreaction PCR isoneofthemostwidelyusedamplificationmethodsforGMOscreeningdetection 3 theneedfordelicateequipmentandcomplicatedprocedureslimittheuseofPCRamplificationinpoint of useandfieldsettings Rapid specific andhighlyeffectivemethodsforidentifyingthepresenceofGMOsinfoodandfeedareimportantandnecessary 4 ThemostfrequentlyusedmethodfordetectingGMOmaterialisscreeningfortheCaMV 35Spromoter P 35S fromthecauliflowermosaicvirus CaMV andthe3 non translatedregionofthenopalinesynthasegene T nos fromAgrobacteriummefaciens 11 Inthiswork wedescribetheinitialdevelopmentofareal timeRPAassaytodetectP 35SandT nossequencesforpurposesofGMOscreeningandetection 2 2 SensitivityoftheRPAAssays 2 3 ApplicationtoPracticalSampleAnalysis 3 1 Materials3 2 ExtractionofGenomicDNA3 3 OligonucleotidePrimersandProbesRPArealtimefluorescentassaysincludeaforwardprimer areverseprimer andaprobe 3 4 RPAAssaysRPAreactionswereperformedinatotalvolumeof50 LusingaTwistAmpExokit TwistDX Cambridge UK 29 5 LofTwistAmprehydrationbuffer 420nMeachRPAprimer 120nMRPAprobe 14mMmagnesiumacetate and1 LofgenomicDNA mixfreeze driedreactiontubeaddMagnesiumacetateandrehydratedmaterialTwistatubescannerdevice 39 Cfor15 25min Fluorescencemeasurementsweretakenevery20s Aprobitregression 3 ExperimentalSection 4 ConclusionsInthisresearch wehavedevelopedarapidreal timeRPAte