Developmentofrecombinasepolymeraseamplifica4onassaystorapidlydetectPhytophthoraspeciesonplantsamples

T.D.Miles1,F.N.Mar4n2,M.Coffey31CaliforniaStateUniversity,MontereyBay,Seaside,CA;2USDA-ARS,CropImprovementandProtecEonResearchUnit,Salinas,CAUSA;3Universityof

California,Riverside,CA.*frank.marEn@ars.usda.gov

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