RPA—PCR技术的革命

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ClinicalFoodsafetyAgriculturalBloodbankscreeningEnvironmentalAnimalhealthRecombinasePolymeraseAmplification(RPA)ofCaMV-35SPromoterandnosTerminatorforRapidDetectionofGeneticallyModifiedCropsChaoXu1,†,LiangLi1,2,†,WujunJin1,2andYusongWan1,2,*1BiotechnologyResearchInstitute,ChineseAcademyofAgriculturalSciences,Beijing100081,China;E-Mails:[e-mail](C.X.);[e-mail](L.L.);[e-mail](W.J.)2InspectionandTestingCenterforEnvironmentalRiskAssessmentofGeneticModifiedPlant-RelatedMicroorganisms(Beijing),MinistryofAgriculture,Beijing100081,China1.IntroductionTheInternationalServicefortheAcquisitionofAgri-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.SensitivityoftheRPAAssays2.3.ApplicationtoPracticalSampleAnalysis•3.1.Materials•3.2.ExtractionofGenomicDNA•3.3.OligonucleotidePrimersandProbesRPArealtimefluorescentassaysincludeaforwardprimer,areverseprimer,andaprobe.3.4.RPAAssaysRPAreactionswereperformedinatotalvolumeof50μLusingaTwistAmpExokit(TwistDX,Cambridge,UK),29.5μLofTwistAmprehydrationbuffer,420nMeachRPAprimer,120nMRPAprobe,14mMmagnesiumacetate,and1μLofgenomicDNA.mixfreeze-driedreactiontubeaddMagnesiumacetateandrehydratedmaterialTwistatubescannerdevice(39°Cfor15–25min)Fluorescencemeasurementsweretakenevery20s,Aprobitregression3.ExperimentalSection4.ConclusionsInthisresearch,wehavedevelopedarapidreal-timeRPAtechniqueforthedetectionofP-35SandT-nosregulatoryelements,whicharewidelyemployedinGMcrops.ThisnovelmethodcanbeeasilyadaptedtoothertargetgenesforGMOdetection.