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    Use of a Multiplex RT-PCR Assay for

    Simultaneous Detection of the North American Genotype Porcine Reproductive

    and Respiratory Syndrome Virus,Swine Influenza Virus and Japanese Encephalitis

    Virus

    AgriculturalSciencesinChina

    2010,9(7):10501057

    Availableonlineatwwwsciencedirectcom

    |^

    :ScienceDireclbcienceirect辨辨July2010

    UseofaMultiplexRT.PCRAssayforSimultaneousDetectionoftheNorth AmericanGenotypePorcineReproductiveandRespiratorySyndromeVirus, SwineInfluenzaVirusandJapaneseEncephalitisVirus

    CHENHongying*,WEIZhanyong*,ZHANGHongying,LOXiao

    li,ZHENGLan.1an,CUIBaoan,LIUJin

    peng,ZHUQianleiandWANGZixin

    CollegeofAnimalHusbandryandVeterinary,HenanAgriculturalUniversity,Zhengzhou450002.PRChina

    Abstract

    Amultiplexreversetranscriptase

    polymerasechainreaction(multiplexRT-PCR)assaywasdevelopedandsubsequently evaluatedforitsefficacyinthedetectionofmultipleviralinfectionssimultaneously.inswine.Specificprimersforeachof

    the3RNAviruses,NorthAmericangenotypeporcinereproductiveandrespiratorysyndromevirus,Japaneseencephalitis

    virus,andswineinfluenzavirus,wereusedinthetestingprocedure.Theassaywasshowntobehighlysensitivebecause

    itcoulddetectaslittleas10

    ngofeachoftherespectiveampliconsinasinglesamplecontainingacompositeofall3 viruses.Theassaywasalsoeffectiveindetectingoneormoreofthesamevirusesinvariouscombinationsinspecimens.

    includinglymphnodes,lungs,spleens,andtonsils,collectedfromclinicallyillpigsandinspleenspecimenscollected

    fromabortedpigfetuses.TheresultsfromthemultiplexRT-PCRwereconfirmedbyvirusisolation.Therelativeefficiency

    (comparedtotheefficiencyofseparateassaysforeachvirus)andapparentsensitivityofthemultiplexRT-PCRmethod

    showthatthismethodhaspotentialforapplicationinroutinemoleculardiagnosticprocedures.

    Keywords:Japaneseencephalitisvirus,multiplexRT-PCR,porcinereproductiveandrespiratorysyndromevirus,swine

    influenzavirus

    INTRODUCTfON

    Porcinereproductiveandrespiratorysyndrome

    (PRRS).whichemergedinNorthAmericaandEu

    ropesimultaneouslyintheIate1980sfWensvoort

    Pf,.1991:Collinseta1.1992).ischaracterizedby

    reproductivefailureinSOWSandrespiratorydisease

    inpiglets(Goyal1993).Thecausativeagentisthe

    porcinereproductiveandrespiratorysyndromevirus

    (PRRSV),amemberofthearterivirusesagroupof

    small,enveloped,positive.strandRNAviruses.

    PRRSVisolatescanbeclassifiedinto2distinct

    genotypes,NorthAmerican(NA)andEuropean(EU), withVR2332andLelystadvirus(LV)beingthepro

    totypevirusesofthe2genotypes,respectively.To date.allthePRRSVstrainsisolatedinChinahavebeen characterizedastheNAgenotype.PRRSVinfections arecharacterizedbyimmunosuppression,whichre

    suitsincombinedinfectionsorsecondaryinfections withtheswineinfluenzavirus(SIV),porcinepseudo

    rabiesvirusandporcinecircovirustype2.vanReeth P,.fl996)foundthattheclinicaleffectsofPRRSV wereexacerbatedinconcurrentinfectionwithSIV. Althoughvarioussubtypesofjnfluenzaviruseshave beenreportedtoinfectswinepopulationsinvarious countries.H1N1HlN2andH3N2havebeenidenti. fiedjnallcountries.

    Received18November.2009Accepted3February.20lO cHENHongying,PhD,Te1:+8637163554361,E

    mail:chhy927@126.com;correpondenceCUIBaoan,Professor,_reI:+86371

    63558878,Fax:+8637163558878,

    Ema1l:baoancui@henaueducn

    Theseauthorscontributedequallytothisstudy @2010CAASAIJnghtsrvedPublishealbyElsevierLtd doJ:101016S1671.2927(09)60189-9

    

    UseofaMuhiplexR%PC

    

    RAssayforSimultaneousDetectionoftheNorthAmericanGenotypePorcineReproductiv(:

    1051

    Underthetypicalconditionsofintensiveswine farming,theanimalsmayoftenshowsimultaneousin.

    fectionwith2ormoreviralpathogens(Ogawaeta1. 2009).Owingtothevariableclinicalsignsofmultiple viralinfectionsandtheconsequentdifficultyinthese

    lectionofthemostappropriatetestingmethod. ade.

    finitivediagnosisinthepresenceofmultipleinfections isoftendifficult.Inseveralpreviousstudies. multiplex

    PCR(mPCR)(EdwardandGibbs1994)hasbeenused toidentifysimultaneouslyanddifferentiatemultiplevi

    rusesinasinglesampleonthebasisoftheamplicon sizes(BelfikandThordn2001:Belfik2005).Thisabil

    ityofmPCRcanfacilitatethescreeningof"panelsof pathogens"inasampleinwhichthetargetedorgan

    ismsaregroupedonthebasisoftheclinicalsymptoms andrelevance(BelfikandThe%2001).InMay2006,a highlypathogenicPRRSVthatcausescontinuoushigh feverandahighproportionofdeathsinvaccinatedpigs OfallagesemergedandisprevalentinmainlandChina sincethen.Porcinereproductivedisorderscausedby otherpathogenslikeSIVandJapaneseencephalitisvi

    rus(JEV)havealsobeenreportedoccasional1Y. Therefore,wedevelopedandevaluatedamultiplexre. versetranscriptasepolymerasechainreaction(multiplex RT-PCR)assayforthesimultaneousdetectionofnucleic acidsfrom3RNAswineviruses,namely,PRRSV,SIV and.1FV

    MATERlALSANDMETHODS

    Virusesandcells

    InOctober2001.aclinicalisolateofSIVwasobtained

fromoneamongagroupofapproximatelyl6wkold

    pigsinHenanProvince,China,showingslowgrowth, coughanddyspnea.Theisolatewaspropagatedinwhite Leghomspecific--pathogen-?free(SPF)chickenembryos andidentifiedasH3N2SIVA/Swine/Henan/703/2001

    (H3N2)].AnotherclinicalisolateofSIVwasisolatedi11 December2005fromoneamongagroupofapproxi

    mately12_.wk--oldpigsinHenanshowingweightloss andcoughing.TheisolatewasidentifiedasH1N1SIV [A/Swine/Henan/407/2005(H1N1)1.TheH1N2SIV [A/Swine/Hebei/1/2006(HIN2)1isolatewasgiftedby Dr.QiaoChuanling(HarbinVeterinaryResearchInstitute, ChineseAcademyofAgriculturalSciences,China).The JEV53-SstrainwaspurchasedfromtheBei..jingOrdi naryMicrobiologyStrainStoreCenter,Beijing,China, andwaspropagatedinthePKl5porcinekidneycell

    Iine.ThePRRSVVR-2332strainwasgiftedbvDrYang Hanchun(ChinaAgnculturalUniversity,China)andwas propagatedjntheMarcl45cellline.Theseviruseswere

    usedasstandardvirusesforthemultiplexRT.PCRand maintainedat70.Cuntilanalysis.UninfectedPK.15. Marc145cellline,allantoicfluids.andclassica1swine fevervirus(CSFV)were

    assays.

    Clinicalspecimens

    alsousedinthespecificity

    FromJune2007toSeptember2008,23clinical specimens,includingthoseoflymphnodes,

    tonsils,

    lungs,colons,duodenums,jejunums,hearts,kidneys,

    livers,spleens,andgonads,werecollectedfrom234. 8wkoldsickpigletsin8loca1farmsand6aborted fetusesfromdifferentabortioncases.Thesamples wereobtainedfromtheVeterinaryMedica1Teaching Hospital,CollegeofVeterinaryMedicine,HenanAgri

    culturalUniversity,China.

    Nucleicacidextractionandreversetranscription ViraRNAwasextractedfrom200.uLaliquotsof samples[10%tissuehomogenatesinphosphatebuff- eredsaline(PBS),whichwerepreparedfrom1gof tissueorvirusinfectedcellculturesupernatant]using acommercialtestkit(QIAampRNAMiniKit,Qiagen, Hilden,Germany)accordingtothemanufacturer's instructions;theRNAextractswerethenstoredat

    20.Cuntilanalysis.

    TheRTreactionwasperformedusing20.uL

    volumes;thereactionmixtureineachvolumecon. tained5xstrandbuffer.25mmolL1ofeach

    deoxynucle0sidetriphosphate(dNTP,AmershamBio. sciencesCorp.,Piscataway,NJ,USA),2.5Uof RNaseinhibitor(PromegaCorporation,Madison,WI, USA),50pmolmLrandomhexamers.Moloney

    murine1eukemiavirus(MMLV1reversetranscriptase (InvitrogenLifeTechnologies,Carlsbad,CA,USA), 5LOfeachRNA,anddiethyIpyrocarbonate

    (DEPC)water.RTwasperformedat42oCfor60

    rainandat75.Cfor10rain.

    ~2010,CAASAtlhghtsreservedPuNishedbyElsevierLtd

1052CHENHongyingetal

    Primerdesign

    ThenucleoproteinfNP)nucleotidesequencesofthe SIVstrains/iso1ateswereretrievedfromGenBankand alignedusingtheDNAStarsoftwarefDNAStarInc.. Madison,WI,USA).Theprimerswereselectedusing thePrimerPremiersoftwarerver.5.0)andwerebased onahighlyconservedsequencewithintheNPregion oftheSIVgenome.AprimerpairspecifictotheD geneofPRRSVwasdesignedtodetectPRRSV.JEV wasdetectedbyidentifyingthenucleotidesequenceof theconservedregionoftheEgene.Theprimerse. quencesforthedetectionofPRRSVandJEVwere obtainedusingthePrimerSelectprogramintheDNAStar 5.0software(DNAStarInc.1.ThePCRprimerpairs foreachtargetgeneandthesizeofeachampliconare summarizedinTable1.

    Table1Specificprimerpairsusedtoamplifyeachtargetgene OptimizationofmultiplexPCRconditions

    Thereactionswereoptimizedbyvaryingtheprimer concentrationsofeachtarget.Optimizationwasper

    formedbymethodicalvariationofeachtestparameter understandardPCRconditions.Thetestedprimercon

    centrationsrangedfrom0.5to50pmol,Aprimercon

    centrationof10pmolyieldedoptimumamplification profilesforalltheprimerssets.DifferentMgC1,con

    centrations(1.5-3.5mmolL01wereevaluated,andthe mostefcientcOncentrati0nwasselected.Thean. nealingtemperatureandnumberofcycleswerealso determinedexperimentally.Thebestresultswereob-

    tainedatanannealingtemperatureof52.Cwith32 cycles.

    SingleRT_PCRassays

    ThesingleRT-PCRassayswereperformedusing 25uLvolumes.Thereactionmixturescontainedthe followingreagents:3.0mmolLMgC1,,1×PCRbuffer

    II(5001111110lLKC1and100mmolLTrisHC1,pH8.3),

    200gmolLofeachdNTP,20pmolofeachprimer, 2LcDNA,and2.5UofAmpliTaqGold(Applied Biosystems,FosterCiCA,USA).Afteroptimization, thefollowingstandardthermocyclerprotocolswere used:(1)ForPRRSV,thecyclingprotocolconsisted ofaninitialdenaturationat94.Cfor3min;25cycles ofdenaturationat94.Cfor30s.annealingat55.C f0r30s.andextensionat72.Cf0r30s:andafinal extensionat72.Cfor10min;(2)forSIV,thecycling protoco1consistedofaninitialdenaturationat96.C f0r3min;25cyclesofdenaturationat95.Cfor30s. annealingat50.Cfor30s.andextensionat72.Cfor 30s:andafinalextensionat72.Cforl0min;(31for JEV.thecyclingprotocolconsistedofaninitialdena

    turationat94.Cfor3min;25cyclesofdenaturation at94.Cfor30s.annealingat50.Cf0r30s.andex. tensionat72.Cf0r30s:andafinalextensionat72.C f0r10min.

    ThePCRamplificationproductswereanalyzed byagarosegelelectrophoresisusing2%agarose stainedwithethidiumbromideandvisualizedun. derUVlight.

    MultiplexRT_PCRreaction

    ThemultiplexRT.PCRconsistsofa2.stepprocedure involvingRTandPCRamplification.TheRTreaction wasperformedusing5LLLOfthethreeRNAmixturein 20uLvolumes.ThemultiplexRT.PCRreactionwas performedusing25uLsamplemixturesintheGene

    AmpPCRKitandAmpliTaqGold.Thereactionmix. turecontained2.5mmolLMgC1.1×PCRbufferII

    (500mmolL0KClandl00inmolL0TrisHCI,PH8.31, 200gmolL0ofeachdNTP.10pmolofeachprimer. and2.5UofAmptiTaqGold.Thismixturewasadded totheRTreactiontubesalongwith100ngoftemplate andDEPC.water.Thecyclingprotocolconsistedof aninitialdenaturationat94.Cfor5min;32cyclesof denaturationat94.Cfor30sannealingat52.Cf0r30s. andextensionat72.Cfor1min;andafinalextension at72.Cf0r10min.Negativecontrolswereusedfor eachtest.AgarosegelelectroDhoresiswasusedtode. tectthemultiplexRT-PCRproducts.

    @2010CAASAllrightsreservedPublishedbyElsevierLtd UseofaMultiplexRT-PCRAssayforSimultaneousDetectionoftheNorthAmericanGenoty

    pePorcineReproductive1053

    Sensitivityandspecificityofsingleandmultiplex RT_PCRassays

    ThesensitivityofeachsingleRT-PCRhasbeenprevl ouslyreported(Schorreta1.1994;Suarezeta1.1994; ParanjpeandBanerjee1998).Thesensitivitiesofthe multiplexRT-PCRandthecorrespondingsingleRT- PCRswerecomparedusingserial10folddilutionsof

    selectedspikedsamplescontainingallthetargetviruses. Thespecificityoftheprimerpairforeachviruswas

    analyzedusingsinglePCR.Thespecificityofan ampliconcorrespondingtoeachtargetwasconfirmed bycloningDNAintothepGEMTEasyvector

    (Promega,USA)andsequencingusinganautomatic DNAsequencer(ABI377;PEAppliedBiosystems,

    USA).ThespecificityofthemultiplexRT-PCRwas assessedintheclinicalspecimen.Thespecificityof themultiplexRT-PCRwasextendedtotheCSF uninfectedPK15,Marc-145cellline,andallantoic fluids.

    Virusisolation

    ToverifytheresultsofthemultiplexPCRassays,vi

    ruseswereisolatedfromallthepositivesamples.The PRRSVwasisolatedusingpreviouslydescribedmeth

    ods(Collinseta1.1992).

    TheSIVwasisolatedbyinoculatingthepositive samplesforthedetection0fSIVintol1d.oldSPF

    embryonatedeggs.Wholesamplehomogenateswere

    diluted(1:5)inPBScontaining100UmL.penicillinG and100mgmLstreptomycinsulfate.Eggswerein

    oculatedwithO.2mLofinoculumintheallantoiccavity. with4eggsperhomogenatesample.Theeggswere candledat24hpostinoculation,anddeadeggswere

    discarded.Theallantoicfluidwasharvestedfromeach eggat72hpostinoculationandwasassayedforSIV. ThepresenceofSIVwasdeterminedusingahemag

    glutinationassay.Equalvolumesofallantoicfluidand 0.5%chickenredbloodcellsweremixedtogetherin V-bottommicrotiterplates.Thepresenceofhemag. glutinationwasassessedafterincubationfor40minat

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