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-3locatedbetween306and329bp,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
1916
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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