RPA-PCR技術(shù)的革命課件

RPA——PCR技術(shù)的革命什么是RPA為什么說RPA是一場(chǎng)技術(shù)革命應(yīng)用及相關(guān)1精選2021版課件什么是RPA？自PCR技術(shù)誕生以來已經(jīng)有三十年了。從經(jīng)典PCR、實(shí)時(shí)定量PCR再到現(xiàn)在的數(shù)字PCR，這一技術(shù)在不斷蛻變卻從未淡出我們的視野。RPA（RecombinasePolymeraseAmplification）

全稱重組酶聚合酶擴(kuò)增技術(shù)，被稱為可以替代PCR的核酸檢測(cè)技術(shù)。2精選2021版課件RPA技術(shù)主要依賴于三種酶：能結(jié)合單鏈核酸(寡核苷酸引物)的重組酶單鏈DNA結(jié)合蛋白(SSB)鏈置換DNA聚合酶。這三種酶的混合物在常溫下也有活性，最佳反應(yīng)溫度在37°C左右。3精選2021版課件RPA的原理4精選2021版課件5精選2021版課件PrevioulyestablishedRPAConditions,20066精選2021版課件7精選2021版課件8精選2021版課件9精選2021版課件10精選2021版課件引物設(shè)計(jì)RPA分析的關(guān)鍵在于擴(kuò)增引物和探針的設(shè)計(jì)。PCR引物多半是不適用的，因?yàn)镽PA引物比一般PCR引物長，通常需要達(dá)到30-38個(gè)堿基。引物過短會(huì)降低重組率，影響擴(kuò)增速度和檢測(cè)靈敏度。在設(shè)計(jì)RPA引物時(shí)，變性溫度不再是影響擴(kuò)增引物的關(guān)鍵因素。RPA的引物和探針設(shè)計(jì)不像傳統(tǒng)PCR那樣成熟，用戶需要自己摸條件進(jìn)行優(yōu)化。11精選2021版課件Speed10to15minutedetectiontimeSensitivitySinglemoleculeCostCheapaccessaslittleornohardwareisrequiredSimplicityStabilisedreactionformat,minimalsamplepreparationRobustnessTosamplecontaminantsandtemperaturefluctuationsPortabilityHandheldinstrumentationordisposabletestformat為什么說RPA是一場(chǎng)技術(shù)革命12精選2021版課件RPA具體應(yīng)用舉例ClinicalFoodsafetyAgriculturalBloodbankscreeningEnvironmentalAnimalhealth13精選2021版課件RecombinasePolymeraseAmplification(RPA)ofCaMV-35S

PromoterandnosTerminatorforRapidDetectionof

GeneticallyModifiedCropsChaoXu1,?,LiangLi1,2,?,WujunJin1,2andYusongWan1,2,*1BiotechnologyResearchInstitute,ChineseAcademyofAgriculturalSciences,Beijing100081,China;E-Mails:xuchao1667@163.com(C.X.);liliang@(L.L.);jinwujun@(W.J.)2InspectionandTestingCenterforEnvironmentalRiskAssessmentofGeneticModifiedPlant-RelatedMicroorganisms(Beijing),MinistryofAgriculture,Beijing100081,China14精選2021版課件1.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.15精選2021版課件16精選2021版課件2.2.SensitivityoftheRPAAssays17精選2021版課件18精選2021版課件2.3.ApplicationtoPracticalSampleAnalysis19精選2021版課件3.1.Materials3.2.ExtractionofGenomicDNA3.3.OligonucleotidePrimersandProbesRPArealtimefluorescentassaysincludeaforwardprimer,areverseprimer,andaprobe.3.4.RPAAssays

RPAreactionswereperformedinatotalvolumeof50μLusingaTwistAmpExokit(TwistDX,Cambridge,UK),29.5μLofTwistAmprehydrationbuffer,420nMeachRPAprimer,120nMRPAprobe,14mMmagnesiumacetate,and1μLofgenomicDNA.mixfreeze-driedreactiontubeaddMagnesiumacetateandrehydratedmaterialTwistatubescannerdevice(39°Cfor15–25min)Fluorescencemeasurementsweretakenevery20s,Aprobitregression3.ExperimentalSection20精選2021版課件4.ConclusionsInthisresearch,wehavedevelopedarapidreal-timeRPAtechni