Developmentofrecombinasepolymeraseamplifica4onassaystorapidlydetectPhytophthoraspeciesonplantsamples
T.D.Miles1,F.N.Mar4n2,M.Coffey31CaliforniaStateUniversity,MontereyBay,Seaside,CA;2USDA-ARS,CropImprovementandProtecEonResearchUnit,Salinas,CAUSA;3Universityof
California,Riverside,CA.*[email protected]
AbstractFluorometric recombinase polymerase amplificaEon (RPA) assays for the genus Phytophthorahave been developed that provide a simple and rapid method to detect the pathogen(Phytopathology105:265-278).Theseassaysareextremelytoleranttowardsinhibitorspresentinmany plant extracts, thereby simplifying DNA extracEon procedures. RPA assays have beendevelopedforPhytophthoragenusspecificdetecEon,species-specificassaysfor9taxaincludingP. ramorum andP.kernoviae, andaplant internal control.Assayswerevalidated for specificityusingDNAextractedfrommorethan135Phytophthorataxa,22Pythiumspp.,andseveralplantspecies with a sensiEvity of detecEon approaching TaqMan real Eme PCR. The assays werevalidatedwith250+symptomaEcplantfieldsamplesrepresenEngmorethan50hosts.Samplesthat were posiEve using the Phytophthora genus specific RPA test were also posiEve usingTaqManPCRandtradiEonal isolaEontechniques.A technique for thegeneraEonofsequencingtemplates from posiEve samples to confirm species idenEficaEon also was developed. Use ofspecies specificTaqManprobe sequences fordesigning species specificRPAprimersprovidesasystemaEcapproach forassaydevelopment.TheseRPAassayshavebenefitsoverPCRbecausetheyarerapid(completedinasli]leas15minutes),donotrequireDNApurificaEonorextensivetrainingtoconduct,requirelessexpensiveequipmentandcanbecompleteddirectlyinthefieldwithportableequipment.ThePhytophthoragenusspecificassayhasbeenmodifiedforuseinalateral flow device and is currently undergoing validaEon, thereby simplifying the ability tocompletediagnosEcsinthefield.
Conclusions
IntroducEonProblemsfordetec4on• TradiEonalplaEngassaysfromplantsamplescantake4-6weekstogetresults,whichcanbe
toolongwhenplanEngdecisionsneedtobemade.• Enzyme-linkedimmunosorbantassay(ELISA)iscommonlyusedinthedetecEonof
Phytophthoraspp.butitisimportanttonotethatbackgrounddetecEonofsomePythiumspp.mayalsooccur.
• SeveralPCRandqPCRdetecEontechniquescurrentlyexisttodetectPhytophthoraspp.ata
genusandspeciesspecificlevelbutallrequiresometypeofDNAextracEonandwelltrainedtechnicalsupporttoperformtheassays.
• Asimple,rapid,reliableandsensiEvetechniquesuchasisothermalamplificaEonwouldbe
extremelybeneficialforsmalldiagnosEclabs,regulatoryagenciesandfieldapplicaEons.Whatisisothermalamplifica4on?• TheabilitytoamplifyDNAwithouttheaidofathermocyclingapparatus• SeveraltypesincludingHelicasedependentamplificaEon(HDA),Loopmediatedisothermal
amplificaEon(LAMP)andrecombinasepolymeraseamplificaEon(RPA)• Verytolerantofinhibitors,soatradiEonalDNAextracEonisnotrequired• Cangrindaplantsampleandhaveresultswithin10-20minutes• TheuseoffluorescentlylabeledprobesallowsformulEplexing• Resultscanbereadonmanydifferentplaeorms,someofwhicharefieldportableObjec4ves1) DevelopaRPAPhytophthoragenus-specificdetecEonassay2) IdenEfycompaEblecrudeEssueextracEonbuffersfortheRPAsystem3) Developasystemtoconstructspecies-specificRPAmarkers,4) DevelopamethodtoconfirmtheidenEficaEonofthespeciespresentinaposiEveRPA
methodbyDNAsequencing5) DeveloparapidfieldportablediagnosEcassaythatcouldbeuseddirectlyatthepointof
samplecollecEon
References/AcknowledgementsBilodeau,G.J.,MarEn,F.N.,Coffey,M.andBlomquist,C.2014.DevelopmentofamulEplexassay forgenus
and species-specific detecEon of Phytophthora based differences in mitochondrial gene order.Phytopathology104:733-748.
Bilodeau,G.J.Koike,S.T.,Uribe,P.,andMarEn,F.N.2012.DevelopmentofanAssayforRapidDetecEonandQuanEficaEonofVer5cilliumdahliaeinSoil.Phytopathology102:331-343.
MarEn,F.N.,Abad,Z.G.,Balci,Y.andIvors,K..2012.IdenEficaEonanddetecEonofPhytophthora:reviewingourprogress,idenEfyingourneeds.PlantDis.96:1080-1103.
SchwartzburgK.,HartzogH.,LandryC.,Rogers,J.,Randall-SchadelB.,2009.PrioriEzaEonofPhytophthoraofconcerntoUnitedStates
TheauthorsgratefullyacknowledgefundingfromtheCaliforniaDepartmentofFoodandAgriculture-2012SpecialtyCropBlockGrantProgramandtheCaliforniaAvocadoCommission.WewouldalsoliketothankMa]hewForrest(TwistDxInc.)foradviceindevelopingthenestedandsequencingtechniquesusingRPA.AddiEonally,theauthorswouldliketothankPaulTooley(USDA-ARS)andGuillaumeBilodeau(CanadianFoodInspecEonAgency)forcriEcalreadingofthemanuscriptandforhelpfuldiscussions.
1) ThedescribedRPAtechniquesshouldhaveasignificantimpactonourabilitytorapidlydetectPhytophthoradirectlyinthefieldandmakemanagementdecisionswithin20minutesofcollecEngaplantsample
2) DuetothefactthatPCRtechnologiescanbeeasilytransferredtotheRPAplaGormitispossiblethatnewassaysforotherspeciesortaxaofplantpathogenscouldbequicklydeveloped
3) SamplescanbesequencesfollowinganestedPCRreacEonswhichallowsfortheconfirma4onofaposi4vedetec4on.
Species Primersrequired
Size(bp) Last6bases Uniquebases GC
P.cactorum 1 36 ATGTAA 11 11%
P.cinnamomi 1 30 GATAAT 17 23%
P.fragariae* 2 31 ATTACG 16 19%
P.kernoviae 15 33 TCACAG 22 15%
P.rubi 2 35 TCTATT 20 25%
P.sansomeana** 2 35 ATAATA 19 14%
P.sojae** 10 29 TATCAA 19 17%
P.ramorum 1 30 TAACGT 17 37%
Primer1 Primer2RPAProbe
TaqManPCRversusRPA
ResultsSensi4vityandspecificity• Validatedallthreetestsonover106Phytophthoraspecies,22Pythiumspeciesanda
widerangeofplantspecies• IdenEfiedreliablebuffersforgrindingplantEssuesamples• TestedsensiEvitywithseveralPhytophthoraspecies• TestedtheeffectofplantmaterialFieldvalida4on• Testedonmanydifferenthostsincludingstrawberry,raspberry,avocado,citrus,bay
laurelandseveralornamentalplants• 222symptomaEcsamples(15counEesinCalifornia)• ValidatedwithReal-EmePCRamplificaEon,sequencingandtradiEonalisolaEonon
selecEvemedia• IdenEfiedseveralPhytophthoraspeciesincludingP.cactorum,P.cinnamomi,P.citricola,
P.citrophthora,P.nicotainae,P.ramorum,P.rubi,P.syringae• ForregulatorypurposesamethodtoconfirmaposiEveusinganestedPCRtechnique
followedwithsequencingwasalsodevelopedandvalidated
Specific'RPA'reverse'primers'AddiEonalspeciesspecificRPAprimers
Fig.1.GraphicalrepresentaEonofTaqManPCRdetecEonsystems(A)andrecombinasepolymeraseamplificaEon(RPA)markersystems(B).
Fig.2.Mitochondriallociusedinthisstudyforthegenus-specificdetecEonofPhytophthora(A),thePhytophthoraspecies-specificassays(P.kernoviaeandP.ramorum)(B)andotherdevelopedspeciesspecificmarkers(Table1).AlsodenotedisthelocaEonofprimersandprobesusedinRPAdetecEonandthelocaEonofnestedPCRandsequencingprimersusedintheconfirmaEonofaposiEveproduct.
Fig.3.ThePhytophthoragenus-specificassay(A)andtheP.ramorumspecies-specificassay(C)rangingfrom2ngto200fg.ThelogoftheiniEalDNAquanEtyofP.ramorumagainsttheonsetofamplificaEon,wherethePhytophthoragenus-specificassayandP.ramorumspecies-specificassaysaredenotedbyclosedandopencircles,respecEvely(B).ThelogoftheiniEalDNAquanEtyofP.ramorumagainstthelogoftheonsetofamplificaEonminustheagitaEonstep(OT),wherethePhytophthoragenus-specificassayandP.ramorumspecies-specificassayaredenotedbyclosedandopencircles,respecEvely(D).
A B
Table1.SomecharacterisEcsofspecies-specificreverseprimersfortheatp9-nad9locus.
Fig.4.AbilitytoamplifyandsequenceRPAproductsfrombothlociaperaRPAdetecEon.
A B
Fig.5.ReacEonscanbereadfluorometricallyonavarietyofdevices(A-D)andcanbeadaptedtolateralflowdevicetechnology(E),allofwhichuseextremelycrudeplantsamples(F).
F
E