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Detection

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Detection

    Detection

    ActaOceanologicaSinica2009,Vo1.28,No.2,P.121126

    http://www.hyxb.org.cn

    Email:hyxbe~263.net

    DetectionofProrocentrumdonghaienseusingsandwich

    hybridizati0nintegratedwithnucleaseprotectionassay

    CHENJie,ZHENYu,MITiezhu,YUZhigang.

    College

    .College

    266100

    .College

    China

    ofMarineLifeScience,OceanUniversityofChina,Qingdao266003,China ofEnvironmentalScienceandEngineering,OceanUniversityofChina,Qingdao China

    ofChemistryandChemicalEngineering,OceanUniversityofChina,Qingdao266100 Received25March2008;accepted16June2008

    Abstract

    P

    rocentrumdonghaienseisanimportantharmfulalgaebloomfHAB1causingcreatureinChina's

    seas.andtheconventiona1visualdetectionCallnotcopewithlong-termmonitoringandhigh- throughputsamplingprojects.AnassayforP.donghaiensewithsandwichhybridizationintegrated

    withnucleaseprotectionassavfNPA

    SH1wasestablished.Testswithmixedsamplesandspikedfield

    onesconfirmeditsgoodspecificityandsensitivity.Thecel1numberofP.donghaiensecorrela

ted

    wellwiththeopticaldensity,andtheregressionequationisy=4×10.+0.6949,inwhichzisthe cel1number.andYistheopticaldensity.withr=0.9535.TheseresultsshowthattheNPASH

    methodhasgoodfeasibilityinthedetectionofP.donghaiense.ResultsofNPA

    SHandnlicroscopy

    areexcellentforeachsample.TheNPA

    SHmethodwasasimplewavinquantitativedetectionof P.donghaiense,andthewholeprocesscouldbefinishedinaboutsixhours,whichprovidedane

    w

    approachinhighthroughputsamplingandlong-termmonitoringofP.donghaiense.

    Keywords'-Pr

    rocentrumdonghaiense,ribosomalRNA.S1enzyme.sandwichhybridizationin

    tegratedwithnucleaseprotectionassayfNPASH)

    1Introduction

    Prorocentrumdonghaiense,whichbelongsto Dinophyta,Dinophyceae,Pror0centrOphycidaeand Proroeentrales.isthedominantharmfulalgalbloom (HAB)speciesintheEastChinaSea(Lueta1.,2003). Itwasreportedthat82harmfulalgaebloom(HAB) eventsoccurredinChinain2007.andthetotalarea amountstoaboutl1610km2.IntheEastChinaSea. 60HABeventsoccurredin2007fBulletinforMarine EnvironmentalQualityofChinain2007,2008),and P.donghaiensewasoneofthedominantspecieswhich causedHABsinthisareaSometimestheareaofP. donghaiensebloomcoversover10000km2andthe HABsdestroyedthemarineecosystem.causingenor. mouslossinaquaculturefZhouandYu,20071.Soitis veryimportantfortheresearchonnlarineecosystem, especiallyonmonitoringofHABstodetectthisspecies

    accuratelyandrapidlyandunderstanditspopulation dynamics.

    Thetraditionaldetectionandquantitativeanaly

    sisofHABspeciesisbasedontheobservationofmot

    phologicalcharacteristics,whichis1aborious,tedious, evensubectiveandincorrectinsomecases.Now moreandmorenewtechniqueshavebeendeveloped (Mieta1.,2003).Amongthem,rRNAtargetedsand

    wichhybridizationassay(SHA)isatypicaltechnique inthedetectionofcausativealgaeofHABsfScholin eta1..19961.ButthereactionconditionsofSHA werenotsostrictlycontrolledanditwasdimcult topreventthetargetedRNAmoleculefroIndegrada

    tion.Thatmaybethereasonthatfewspecificprobes havebeendesignedsincethismethodappearedten yearsago(Tyrrelleta1.,2002).Caieta1.(2006)and Zhenf2006)developedthesandwichhybridizationin

    tegratedwithnucleaseprotectionassay(NPASH)on

    thebasisofSHA.ThistechniqueusesS1nuclease. whichonlypreservesperfectlymatchedprobes,tocon

    vertrRNAtoequivalentmatchedstableDNAprobes. IttakestheadvantageofhighcopynumbersofrRNA andmeanwhileovercomesthedrawbackoftheunsta

    bleandunspecificuatureofsandwichhybridization assay.ThistechniquehasachievedSuccessinthe detectionofP.micans,Skeletonemacostatumand Foundationitem:TheNationalHighTechnologyResearchandDevelopmentProgram("863

    "Program)ofChinaundercontract

    No.2006AA09Z178;theNationalNaturalScienceFoundationofChinaundercontractNo40

    706044.

C0rresp0ndingauthor,Email:zhigangyu@ouc.edu.cn

    122CHENJieeta1.ActaOceanologicaSinica2009,Vo1.28,No.2,P.121126

    Phaeocystisglobosa,etc.Theanalysisneedstode

    signspecifico1igonucleotideprobesforeachalga,in

    cludingacaptureprobe,anucleaseprotectionas

    say(NPA)probeandasignalprobe.Thefunction ofcaptureprobesistohybridizewiththetargeted sequences(NPAprobes)onthesolidcarrier.The functionofNPAprobesistohybridizewiththetar

    getedmicroalgaerRNAandtransfersthesequence andquantitativeinformationasamedia.Thesignal probeslabeledwithfluoresceinat3endhybridizewith theNPAprobesarequantifiedwithenzymemediated

    colorreaction.Thesignalintensitycorrelatestothe abundanceofthetargetspecieintheoriginalsan!

    ples(Zhen,2006).Becausethereisrelativelystable amountofrRNAinacel1.cellnumberscanbecal

    culatedfromit.Thustheinicroalgaecanbedetected quantitatively.

    Inthisstudy.asetofspecificprobesforP.dong

    haiensewasdesigned,andtheNPASHwasappliedto

    detectthismicroalgaqualitativelyandquantitatively. 2Materialsandmethods

    2.1Aaecultures

    Ninespeciesofmicroalgaewereusedinthis study,whichwereP.donghaiense,Gymnodiniumsp., P.minimum,P.micans,P.triestinum,G,mikimo. toiChaetoceroscurviestusSkeletonemacostatumand Phaeocystisgloboaa.ThespecieswerefromthePhy

    toplanktonTaxonomyandEcologyLaboratoryofthe

MarineLjfeCollege.OceanUniversityofChina.Qing

    dao,China.Thealgawasculturedaxenicallyinf/2 mediumat18.Cona12:12hlight:darkcyclewitha photonfluxdensityof4000lx.

    2.2R?Aextraction

    RNAextractionwasperformedaccordingtothe protocoldescribedbyZhen(2006).

    2.3rRNAprimersdesign

    ThroughBLASTnprogramonline,homologsto thesequencesofsmallsubunitrRNA(SSUrRNA1and

    large-subunitrRNA(LSUrRNA)inmicroalgaewere found.PrimersweredesignedwithPrimerPremier5.0 softwarewithinthemostconservedregionsi11theSSU rRNAandLSUrRNA.Sixprimerswerepresentedin Table1.

    2.4RT-PCRamplifications,cloneandsequene

    ing

    RTPCRamplificationswereperformedusing primersinTable1andcommercialkitfOneStepRNA PcRKit,TaKaRa,Biotechnology,Dalian,China). Al1PCRproductswereseparatedbyelectroDhoresis ona1%agargels.ThetargetDNAbandswererecov

    eredandpurified.theninsertedintoPMD18Tvec

    torandexpressedbytransformingE,coliDH5corn

    petentcells.Thepositivecloneswereidentifiedby doubleenzymedigests,andthensequencedbySan

    gonBiotechn0logiesInc.(Shanghai,China).SSU andLSUrRNAsequencesforP.donghaiensewereob- tainedfAY803743forSSUrRNAsequence.AY833516 f0rLSUrRNAsequence1.

Table1.Primersinthestudy

    2.5Probesdesignandsynthesis

    TherRNAsequenceofP.don.

    qhaiensewasan

    alyzedusingBLASTn,FromtheresultsofBLAST. thedifierentregionsinrRNAfrOlnotheralgaeth mainlyreferredtothosebelongingtothesamegenus butdifierentspecieswerefound.wheretheNPAprobe wasdesigned.ThentheNPAprobewasanalyzed usingBLASTnprogramagaintoconfirmitsspeci

    ficity.Afterthatthecorrespondingcaptureprobe andsignalprobeweredesigned.Finallytheoligonu- cleotideprobeswerechemicallysynthesizedbySan

    gonBi0technologiesInc.(Shanghai,China)(Table2). Thecaptureprobewaslabeledwithbiotinandcorn- plementarytothe3terminalregionofNPAprobe andthesignalonewaslabeledwithfluoresceinat3 endandcomplementarytothe5terminalregionof NPAprobe.

    CHENJieeta1.ActaOceanologicaSinica2009,Vo1.28,No.2,P.121126

    Table2.SequencesofprobesforP.donghaienseinthisstudy 2.6NPA.sH

    ByusingNPASHtodetectP.donghaiense.many

    factorscouldinfluencethefinalresults.suchase伍一

    ciencyofRNAextraction,hybridizationtemperature, andconcentrationofprobes.Throughmanytests, theseinfluentialfactorswerestudiedandtheappro

    priatestandardswerebuiltupfZhen,2006).Accord

    ingtothesestandardsandresultsfromexperiments, thisstudygottheoptimalconcentrationofeachprobe

    forP.donghaiense.whichwere5nmol/dmforthe captureprobe.50nmol/dmfortheNPAprobeand 5nmol/dm3forthesignalprobe.0nthebasisof them.NPASHwasestablishedandappliedtodetect P.donghaiense.

    TtieNPASHanalysiswascarriedoutaccording toCaieta1.f20061withmodification.Cellsinthe stationarygrowthphaseweretransferredinto1.5ml microcentrifugetubes,collectedbythecentrifugation at12000r/rainfor5min.withtheadditionof1ml1y

    sisbufferafterdiscardingsupernatant,andsonicated at50%dutycycleand450Woutputfor4rain(Ul

    trasonicCellDisrupter.modelJY92II.Ningbokesh

    engInc.,Zhejiang,China)beforefiltrationundervac

    uum.27celllysatefiltrate,3lNPAprobesolu

    tionf500nmol/dm0NPAprobesin1ysisbuffer)and 50"lmineraloilweretransfcrredtoa1.5mlmi

    crocentrifugetubedenaturedat94.Cfor15min. cooledandincubatedat42.Cfor2hforhybridiza- tion,then30lS1nucleasebuffer(60unitsS1nucle. asein1.4mol/dm3sodiumchloride.22.5mmol/dm3 zincsulfate,250mmol/dm3sodiumacetate,pH4.5) fPromega,USA)wasaddedandincubatedfor1h at42.C.andthedigestionreactionwasstoppedby adding150stopsolution62.5mmol/dm.sodium

    hydroxide,30mmol/dm3EDTAand0.5mol/dma phosphatebufferedsaline(PBS),pH7.2].Themix

    turewasdenaturedat94.Cfor15rain,andcooled toroomtemperaturefor5rainforfurtheranalysis.

    Streptavidincoatedmicroplate(PierceBiotechnology, Inc.Rockford.IL.1wascoatedby5nmol/dm3biotin

    1abeledcaptureprobes.andthenwasfilledwith100 "l/wellmixturetreatedbyS1nucleaseandcovered with50ttlmineraloilbeforeincubationat50.C 123

    for1hundergentleshaking(200r/min),andthen washedfivetimeswithPBScontaining0.5%Tween 20onaplatewasher.Next,thewellswerefilled with100"l5nmol/dmsignalprobesinhybridiza- tionbufferf4×SSC,10%formamide,0.02%SDS,PH

    7.2,perwel1.andincubatedat50.Cfor30minun

    dershaking(200r/rain).Afterwashing,1001ofthe antifluoresceinPODfRoche,USA.1:6000dilution inPBS,2%goatserum)wasaddedtoeachwell,incu

    batedat37.Cfor30min.washedfivetimes.Finally, 100lperwellof3,3,5,5'-tetramethylbenzidine (TMB,Sigma,USA)substratewasadded,incubated at37.Cfor15minforcolordevelopmentandthecolor reactionwasstoppedwith50"lof2mol/dmH2SO4 perwellandtheopticaldensity(OD)wasmeasured at450nmand630nmonamicrowellplatereader (modelST360,KHBInc.,Shanghai,China).Theab

    sorbancewasdeterminedat450nmwithreferenceat 630nm.

    3Results

    3.1Specificityofthe0Z90cZeDdeprobes

    Similarnumberofmicroalgaecellsmentioned abovewerecollectedandlysedrespectively.Theneach lysatewasmixedwithNPAprobeforP.donghaiense

    andanalyzedbyNPASHforP.donghaiense.Finally. theODat450nmwithreferenceat630nmwasmea. sured.TheresultshowsthatthesignalfromP.dong

    haiensewasobviouslyhighandtheonesfromother microalgaeweremuchlower(Fig.1).Theresultsin

    dicatethatoligonucleotideprobesforP.donghaiense hadgoodspecificity.

    3.2Standardc~trve

    AcertainnumberofP.donghaiensecellsinthe stationaryphasewerecollected,lysed,andseriallydi

    1utedwith1ysisbufferrespectively.Eachdilutionwas analyzed3timeswithNPASH.TheODat450nm

    withreferenceat630nmwasdrawnagainsttheHum- berofcells,acurvewasobtainedfFig.2).Thelinear partwaschosen,andthestandardcurveofdetecting P.donghaiensewasdrawn.Xaxisindicatedthecell numbersandY.axisindicatedtheopticaldensity.The 124

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    Fig.1.Specificityofthe0ngonucleotideprobesforP.donghaiense

    0O.20.40.60.81.01.2

    Cellnumber/1O

    Fig.2.Theanalysiscurveforthedetectionof P.donghaiense.

    1inearrangewasfrom4.5x103to3.7x10cells.and thelinearregressionequationisy=4x10_..x+0.6949, withr2=0.9535(Fig.3).

    3.3SamplesdetectbyNPASH

    3.3.1Culturedsamplesanalysis

    ThreesamplesofculturedP.donghaiensecellsin thestationaryphasewerecollectedwithdifferentcell Cellnumber/l

    Fig.3.Theregressioncurveforthedetection ofP.donghaiensebyNPASH.

    densities.Eachsamplewascountedwithmicroscopy andNPASHforP,donghaiense,andthetwoenumer

    ationresultswerecompared(Table3).

    Theresultsofcellenumerationusingtwometh- odswereveryclose.Throught-test,therewasno

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