RPA——PCR技术的革命

.,RPAPCR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37C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,andsoybean1.,Althoughthepolymerasechainreaction(PCR)isoneofthemostwidelyusedamplificationmethodsforGMOscreeningdetection3,theneedfordelicateequipmentandcomplicatedprocedureslimittheuseofPCRamplificationinpoint-of-useandfieldsettings.Rapid,specific,andhighlyeffectivemethodsforidentifyingthepresenceofGMOsinfoodandfeedareimportantandnecessary4.,ThemostfrequentlyusedmethodfordetectingGMOmaterialisscreeningfortheCaMV-35Spromoter(P-35S)fromthecauliflowermosaicvirus(CaMV)andthe3non-translatedregionofthenopalinesynthasegene(T-nos)fromAgrobacteriummefaciens11.Inthiswork,wedescribetheinitialdevelopmentofareal-timeRPAassaytodetectP-35SandT-nossequencesforpurposesofGMOscreeningandetection.,.,.,2.2.SensitivityoftheRPAAssays,.,.,2.3.ApplicationtoPracticalSampleAnalysis,.,3.1.Materials3.2.ExtractionofGenomicDNA3.3.OligonucleotidePrimersandProbesRPArealtimefluorescentassaysincludeaforwardprimer,areverseprimer,andaprobe.3.4.RPAAssaysRPAreactionswereperformedinatotalvolumeof50LusingaTwistAmpExokit(TwistDX,Cambridge,UK),29.5LofTwistAmprehydrationbuffer,420nMeachRPAprimer,120nMRPAprobe,14mMmagnesiumacetate,and1LofgenomicDNA.mixfreeze-driedreactiontubeaddMagnesiumacetateandrehydratedmaterialTwistatubescannerdevice(39Cfor1525min)Fluorescencemeasurementsweretakenevery20s,Aprobitregression,3.ExperimentalSection,.,4.ConclusionsInthisresearch,wehavedevelopedarapidreal-timeRPAte