多重PCR的优化

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in5mlofLBbrothandgrownagainat37◦C,150rpmfor3h.Thedilutionsofcultureweremadeatthispointandusedfortemplatepreparation.Templatepreparation

TheLBgrownculturescontaining109c.f.u.werese-riallydilutedwithsteriledeionizedwatertogive105to101cellsper0.5ml.DilutionswereconﬁrmedbyspottingonEMB/SS/TCBSagarplates,respectively.Thedilutedsamplesoftherequiredcellconcentra-tionswerepooledandcombinedinasingletubetogive1.5mloftotalvolume.Themixturesofthesecellswereharvestedbycentrifugationat20000×gfor30min.Thecellpelletswerewashedtwicewith1.0mlofsterilesalinesolution(0.9%NaClindeion-izedwater)andoncewithsteriledeionizedwaterandpelletedat20000×gfor30min.Thepelletsweresuspendedin8.5µlsteriledeionizedwater.InaPCRtube,8.5µlofcellsuspensionwasmixedwith1µl10×TE(100mMTris,5mM1EDTA,pH8.0)and0.5µlproteinaseK(20mgml−);togiveaﬁnalcon-centrationof10µgproteinaseKin1×TE.ThecellswerelysedtoreleasethetargetDNAbyincubatingat37◦Cfor15min,60◦Cfor10minandﬁnally,theproteinaseKwasdenaturedat98◦Cfor10minThelysedsampleswereuseddirectlyastemplateintheM-PCRorkeptfrozenat−20◦Cuntilneeded.Primersused

Thetargetpathogenswereselectedsincetheyareknowntocausewaterandfoodbornediseases.Followingpathogensandtheirtargetlociwereusedinthestudy.E.coliisprimarilyahabi-tantofhumangutandhence,ausefulindica-toroffecalcontamination.PCRprimersthatwereusedinthedetectionofE.colitargetedthelamBlocus(Bejetal.1990).ThereportedprimersBL4910󰀅(5󰀅-CTGATCGAATGGCTGCCAGGCTCC-3)andBR5219(5󰀅-CAACCAGACGATAGTTATCA-CGCA-3󰀅)wereusedtoamplifya309bpfrag-ment.ThefollowinginvAandctxAprimershavebeendesignedusingtheLaserGenesoftware,DNAS-TAR.SalmonellaeoneoftheetiologicalagentsfortyphoidandgastrointestinalinfectionweredetectedwiththeinvAlocus.Thisproteinisacomponentofthesec-independentsecretionapparatus,whichisnecessaryforfullvirulenceofthebacteriumasdemonstratedbyinvitrostudies(Clarketal.1996,Jonesetal.1994).Salmonellaspp.primersweredesignedfromtheinvAgene(Galanetal.1992,

GenBankaccessionno.M90846).PrimerInvAF,5󰀅-CCTGATCGCACTGAATATCGTACTG-3󰀅,islocatedbetween201and225bp,andprimerInvAR,5󰀅-GACCATCACCAATGGTCAGCAGG-3󰀅,islocatedbetween776and799bp;andcouldamplifya598bpproduct.ThectxoperonofVibriospp.waschosenastargetlocusforinclusionintheM-PCRprotocolasitisknowntobeassociatedwithpathogenicity(Shi-raietal.1991).A435bpproductisampliﬁedfromVibriospp.usingtheprimersderivedfromthectxAse-quence(GenBankaccessionno.D30053).TheprimerctxAF-1,5󰀅-CTCAGACGGGATTTGTTAGGCACG-3󰀅locatedbetween306and329bp,andctxAR,5󰀅󰀅-GATCTTGGAGCATTCCCACAACC-3asantisenseprimer,locatedbetween719–741bp,whichcouldam-plifya435bpproduct.AlltheprimerspurchasedinthestudyweresynthesizedbyGibcoBRL,USA.PCRampliﬁcation

ThereactionswereperformedinthePerkinElmerGeneAmpsystem9600.DifferentprogramsusedintheoptimizationoftheM-PCRarelistedinTable1.Thetimerequiredbetweenthetwotemperaturestepsasgradientisdeﬁnedastheramp.Thereactioncon-ditionswereusedasdescribedearlierforduplexPCRforSalmonellaandVibrioinaﬁnalreactionvolumeof25µl(Kapleyetal.2000).

Results

Factorsthatinﬂuencetheampliﬁcationofmulti-targetlocihavebeeninvestigatedwithspeciﬁcreferencetoampliﬁcationofenteropathogensE.coli,SalmonellaandVibriospp.Themajorfactorsinvolvedinproductampliﬁcationarediscussedbelow.Effectofthethermocyclingprogram

Thermocyclingprogramsusedindevelopingthemul-tiplexPCRassaytodetectthreepathogensareshowninTable1.ThePCRproductproﬁlefordifferentther-mocyclingprogramsisshowninFigure1.Templateconcentrationusedinthereactionforalltheprogramswas103c.f.u.Asingle-stepPCRreactionwithanan-nealingtemperatureof55◦Cdidnotamplifyallthreelociandresultedinampliﬁcationofmanynon-speciﬁcproducts(Figure1,lane2).Similarly,byvaryingthetemperaturebetween45–60◦C,nodifferencewasob-served(datanotshown).ProgramB,thedouble-stepprogram,alsodidnotyieldthePCRproductsforthe1915

Table1.DevelopmentoftemperatureprogramsforoptimizationofM-PCR.

ProgramAProgramBStep1Step2ProgramCStep1Step2Step3ProgramDStep1Step2Step3Step4Step5

35cyclesof94◦Cfor60s,45/55/60◦Cfor90sand72◦Cfor60s15cyclesof94◦Cfor30s,45◦Cfor90sand72◦Cfor20s20cyclesof94◦Cfor20s,60◦Cfor45sand72◦Cfor30s

5cyclesof96◦Cfor30s,60◦Cfor60s,72◦Cfor30swithrampof170s5cyclesof95◦Cfor30s,55◦Cfor60s,72◦Cfor30s,withrampof170s20cyclesof94◦Cfor20s,51◦Cfor30s,72◦Cfor45s,witharampof60s5cyclesof95◦Cfor30s,45◦Cfor10s,72◦Cfor10s,witharampof90s5cyclesof95◦Cfor30s,51◦Cfor30s,72◦Cfor10s,witharampof70s5cyclesof95◦Cfor30s,◦Cfor30s,72◦Cfor10s,witharampof60s5cyclesof95◦Cfor30s,57◦Cfor60s,72◦Cfor10s,witharampof50s10cyclesof94◦Cfor10s,60◦Cfor60s,72◦Cfor15s,witharampof80s

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Fig.2.Demonstrationofsensitivityandoptimizationofprimerra-tiosintheM-PCRusingthermocyclingprogramD.Lanes1,7,9and14showthe100bpstandardladderfromGibcoBRL,USA.Lanes2to6demonstratethesensitivityofM-PCRwithtemplateconcentration5derivedfromE.coli,SalmonellaandVibriospp.;lane2,10c.f.u.;lane13,104c.f.u.;lane4,103c.f.u.;lane5,102c.f.u.;lane6,10c.f.u.;lane9,ampliﬁcationproductusing103c.f.u.Pseudomonasputidaastemplate;lanes10to13showthedifferentprimermixingratiostried.Lane10had0.25:0.25:0.25,lane11had0.25:0.5:0.5,lane12had0.25:0.5:0.25andlane13had0.25:1.0:1.0ratiosoflamB:invA:ctxAinµMconcentrationperreaction,respectively.

organismusingthefollowingprimerconcentrations.TheprimermixingratiosusedwereasfollowsinthedifferentlanesseeninFigure2,viz.,lane10had0.25:0.25:0.25,lane11had0.25:0.5:0.5,lane12had0.25:0.5:0.25andlane13had0.25:1.0:1.0ratiosoflamB:invA:ctxAinµMconcentrationperreaction,re-spectively.Allprimerconcentrationstriedshowedtheampliﬁcationofthethreetargetlocibutwithdifferentdegreesofampliﬁcationofeachproduct.

Thepreferenceinampliﬁcationofproductsvarieswiththeprimerconcentrationevenwhenthetargettemplate,asnumberofcellsperreaction,waskeptconstantasshowninFigure2,lanes10–13.ThelamBproductwasobservedtobemoredominantthaninvAandctxAwithalltheprimerratiostried.Equimo-larprimerratiodoesnotamplifyallthree-targetlocievenly,asseeninFigure2,lane10.WhenonlytheconcentrationofinvAprimerswasincreasedto0.5µMtherewasnosigniﬁcantchangeobserved(lane12);italsohadnoeffectonnon-speciﬁcampliﬁcation.Theprimerconcentrationwhichledtothebestpossibleoptionforalltheproductstoamplifyequallyasseeninlane11waslamBat0.25µM;invAandctxAat0.5µM.IncreasingtheconcentrationofinvAandctxAto1µMgaverisetonon-speciﬁcampliﬁcationasshowninlane13.

Thespeciﬁcityoftheprimerswastestedbyusing103

c.f.u.ofPseudomonasputidaasthetemplateandusingtheprimerratiodescribedforlane11.Thisre-actiondidnotgivenon-speciﬁcextensionofprimersevenafterloadingcompletereactionvolume,asshowninFigure2,lane8;itsigniﬁesthattheprimersarespeciﬁctothetargettemplate.Similarly,theadditionofthesamenon-speciﬁctemplatedidnotalterthePCRpatternwhentriedwithbasiccompositionofreactionasusedforlane11(datanotshown).SensitivityoftheM-PCR

Templateconcentrationrangingfrom105to101c.f.u.ofeachorganismwastestedtodeterminethesensi-tivityoftheM-PCR.Targettemplateswereampliﬁedusingtheoptimumprimerconcentrationasdescribedforlane11.TheampliﬁcationproductscanbeseeninFigure2,lanes2to6.Theresultsshowthatthesensitivity1ofthereactionis102c.f.u.Thelanewith10c.f.u.ofeachorganismdidnotshowanyproduct.Thiscouldprobablybeduetothefactthecellscouldnotberecoveredintheinitialprocessoftemplatepreparation.

Discussion

M-PCR,sinceitwasﬁrstreported,hasbeendiscussedindetailregardingtheinteractionofitsreactioncom-ponentstoamplifyoneconservedlocusfromdifferenttemplatesordifferentlocifromthesametemplate(Chamberlainetal.1988,Francketal.1998,Hene-garieuetal.1997,McGregoretal.1996,Tsen&Jian1998,Wangetal.1997).Inthispaperwede-scribetheimportanceofthethermocyclingstepsandroleofmulti-steptemperaturecycles,whichensurethespeciﬁcannealingofprimershavingdifferentmelt-ingtemperatures.ThereactionmixturealsohadtheheterogeneoustemplatederivedthroughasimpletotalDNAextractionprotocol.Wehavedescribedtheor-ganismspeciﬁcPCR,whichhasbeenoptimizedbasedonasinglestepPCRprotocol(Purohitetal.1997).Thesinglestepcycle,whenextendedtoeventwoloci,resultedonlyinsmearingasnon-speciﬁcexten-sion.◦Theannealingtemperaturestriedwere45◦C,55Cand60◦C.AllthreegavethesameresultthatisshowninFigure1,lane2.Theprogramearlierre-portedforSalmonellaandVibrioduplex(Kapleyetal.2000),whichusesathree-stepprogram,alsogaveonlylamBproductwiththicksmearingcoveringfaintctxA

product.Thismaybeduetoadditionaltemplateload,whichreducestheprimerspeciﬁcity(datanotshown).ProgramBwasdesignedwith15cyclesofampliﬁ-cationat45◦C.Theremainingcycleswerecarriedoutat60◦CtoincreasethespeciﬁcityoftheM-PCRasshowninTable1.ThisprogramgaveabandforE.colihiddeninthesmearingduetonon-speciﬁcproducts.Theresultssuggestedthatthedensezoneofnon-speciﬁcproductsbetween300bpto700bpmightbeduetouncontrolledextensionofspeciﬁcallyannealedprimers.Thispromptedustousetherampinthenextprogramdesigned,aswasusedbeforeindu-plexPCR(Kapleyetal.2000).Thethree-stepprogramCwastriedtoaddressthisnon-speciﬁcextension.Thereasoningherewastoreducethenon-speciﬁcsmear-ingbycarryingouttheampliﬁcationoftheﬁrstfewcyclesathighertemperature,whichshouldresultinampliﬁcationofspeciﬁctargetproducts.ThismethoddidamplifythelamBandinvAlocus,howeverthectxAcouldnotbeampliﬁedbecauseeithertheanneal-ingtimeorthetemperaturewasnotoptimuminthismethod.

Aftertryingdifferentcombinationsthebest-ﬁtconditionsresultedinprogramD,whichampliﬁedallthethreeproductswithsensitivityupto102c.f.u.Thedescendingtemperatureforannealingstartingat60◦Cto45◦C,i.e.,step5tostep1failedtoamplifybothinvAandctxA.ProgramDstartedwithﬁvecycleshav-ingannealingtemperatureof45◦Ctoco-amplifythethreetargetedgenes.Theannealingtemperaturewasraisedto51◦Cinstep2,andthereafterthe◦annealingtemperaturewasraisedattherateof3CforeverystepintheﬁvestepsofprogramD.Thesamepro-gramwhenoperatedwithoutconsideringtherampandwiththeusualtimerequiredbetweenthetemperaturestepsbythermocycleryieldedlighterproductbandscoveredwithsmearing.The◦ramp,withslowertem-peraturegradientthan0.3Cs−1gavenon-speciﬁcextensionfortheﬁrstfour◦steps.Thelaststep,how-ever,didrequirea0.15Cs−1gradientsinceituses60◦Casannealingtemperature(datanotshown).Therampprovidestheconditionwherethespeciﬁcprimersstayontothetemplateevenafteratempera-turegradient.Thesameconditionsremovethepoorlyannealedprimers,therebyreducingthemis-primingintheextensioncyclesasshowninFigure2.

PrimerratiosalsoaffectM-PCR,besidesthean-nealingtemperaturesandextensiontime.Initialre-sultsshowedthatusingequimolarconcentrationsof0.25µMprimerswithahighnumberoftemplategavegoodresultswith105c.f.u.(datanotshown).How-1917

ever,theseprimerconcentrationswerenotefﬁcienttoequallyamplifyallthethreePCRproducts3whentemplateconcentrationwasloweredto10,asseeninFigure2,lane10.Byadjustingtheprimerratioasdescribedearlier,wefoundthatevenwithalowtemplateconcentration(c.f.u.103),eachtargetproductwasequallygenerated.Theratioof1.0µMprimersforSalmonellaandVibriogaveadditionalbandswith103andabovetemplatelevels.

TheoptimizationoftemperaturecyclesforM-PCRprotocolinthisstudyyieldedaprotocolforthede-tectionofthreewater-bornepathogens.Thedepletingreservesofcleanandpotablewaterisamajorcon-cernforthedevelopingcountries.AWHOreportin1996statesthatthedevelopingworldfacesahighriskfromcholeraandtyphoidinfections(WHO/EOS/96);hence,wehavechosenSalmonellaandVibrioastar-getorganismswithE.coliasanindicatoroffecalcontamination.WehaveearlierreportedtheduplexampliﬁcationofSalmonellaandVibriousingthephoEprimers.Eventhoughtheseprimershavebeentestedfor132differentSalmonellae,wehavenotincludedtheminthismultiplexreaction,sincetheampliﬁca-tionproduct(365bp)doesnotclearlyresolveonthegelalongwiththelamBproduct(309bp).PrimersspeciﬁcforVibriospp.ampliﬁedaregionofthectxoperonthatencodesforcholeraenterotoxin,amajorvirulencedeterminantofV.cholerae01.PrimerctxAFandctxARgiveanampliﬁcationproductof485bp(Kapleyetal.2000),whilectxAF-1,usedinthisstudy,andctxARgavea435bpproduct.Theuseofeitherprimersetdidnoteffectthesensitivityoftheprogram.Thissuggeststhatthedevelopedthermocyclingproto-colDcanaccommodateminorﬂuctuationsinprimertypes.Forthemultiplexprotocol,wehaveusedthe435bp-ampliﬁcationproductofthectxAgenesinceitisclearlydistinguishablewithlamBandinvAonagarosegels.Acknowledgement

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