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PCR2DGGE,,,(,200092):PCR2DGGEDNA,16SrDNAV3PCR,DGGE(),.,.2,.16SrDNAV3,NCBI(),.,PCR2DGGE.:;;;;:X70311:A:025023301(2004)0620074206:2003212212;:2004202216:(863)(2002AA601320):(1978),,,,CharacteristicsofMunicipalSewageChem2BioflocculationTreatmentProcessbyUsingPCR2DGGETechnologyWANGFeng,FUYi2gang,XIASi2qing,YANGDian2hai(StateKeyLaboratoryofPollutionControlandResourceReuse,SchoolofEnvironmentalScienceandEngineering,TongjiUniversi2ty,Shanghai200092,China)Abstract:Itisreportedthatwithoutcultivation,DNAcouldbedirectlyextractedfromenvironmentalsampleswithmolecularbiologi2calmethods,suchaspolymerasechainreaction(PCR)anddenaturtinggradientgelelectrophoresis(DGGE).Toanalyzethecommuni2tydiversityofactivatedsludgeandbio2filminthemunicipalsewage,workwasdonetodirectlyextrudecrudeDNAfromactivatedsludgeandbio2filmsamples,separateandamplify16SrDNAbyPCRandsequenceitwithDGGE.Theresultsshowthesignificantmicrobecommunitydifferencebetweencultivatedanduncultivatedactivatedsludge.Furtherresearchonthecommunitydiversityoftwodifferentsewagetreatmentprocesseswasdoneandinitialdiscussiononthemicrobialdistributioninthesamereactorandmicrobialstructureindifferentexperimentalconditionswascarriedout.Thesequencesofseveral16SrDNADGGEfragmentsweredeterminedandsomepossiblebacteriawereconfirmedincomparisioninGeneBank(NCBI).TheresultsshowthatthePCR2DGGEtechnologycombinedwithsequencesdeterminationisafeasibleandefficientmethodformicroorganismanalysisinenvironmentalsample.Keywords:PCR;DGGE;communitydiversity;activatedsludge;bio2film.,,.,,.,.PCR2DGGE(PolymeraseChainReaction2DenaturingGradi2entGelElectrophoreses),,DNA,16SrDNA,,.,.,.2.(30min)256200411ENVIRONMENTALSCIENCEVol.25,No.6Nov.,2004转载[1,2].PCR2DGGE,.1111(1).2,,.(),(),3,,.,,12h,(1).2,1.1(50t/d)Fig.1Thepilotplant(50t/d)inShanghaiZhuyuanwastewatertreatmentplant(2)1mL100,1mL[3],120r/min,324d5000r/min20min,DNA..(3)DNA:100mmol/LTris2HCl(pH810)[4],100mmol/L(pH810)[4],115mol/LNaCl,1%CTAB()[4].TE:10mmol/LTris2HCl,pH716[4],1mmol/LEDTA(),pH810.PCR16SrDNAV3PCR(P152ATTACCGCGGCTGCTGG23,P252CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCCCATACGGGAGGCAGCAG23),GCDGGE[4]..MarkerBebcoDNA30002100bpMarker.(4)5000r/min20min,-30DNA.,5000r/min20min,-30DNA.1Table1ThepositionsandprocessconditionsforsamplingAB1123/mgL-1COD320263126NH32N281026102411TP3154112101SS17415660DO211mg/L123117mg/L115mg/L113mg/L123215mg/L212mg/L116mg/LpH71261871361671380mg/L120mg/LPAM015mg/L576中国科技论文在线(1)DNA[68]DNA:1315mLDNA100LK(10mg/mL).,37,225r/min30min.115mL20%SDS()[4],652h,1520min.5000r/min10min,.415mLDNA,015mL20%SDS,,6510min,10min.1,3,./(241),5000r/min10min,;016,1h,9000r/min20min,500LpH716TE.(2)PCR[68]94,2min;94,45s,60,45s,72,90s,30cycles;72,10min.PCR39L,10PCR5L,215mmol/LdNTP1L,10mmol/LP1P21L,Taq1,DNA2L.(3)DNAPCR1%(4)PCR,Parafilm,-70,4h,30LpH716TE,-20.(5)PCRDGGE()30%60%.150V,1TAE[4],60,5h.,DNA.,,30LTEpH716.PCR,(2)..2211DNA8DNA1%(23).SmartViewDNA56kb.,DNAPCR2DGGE.,DNA.1.Marker;21Sample;31Sample;4.Sample;51Sample2ADNAFig.2ExtractedDNAfromSample,,1.Marker;21Sample;31Sample;4.Sample;51Sample3BDNAFig.3ExtractedDNAfromSample,,212DNA16SrDNADNA16SrDNAPCR,1%,DNA220240bp.16SrDNAV3(45).21316SrDNA6PCR(50L),(6)...(6a),(Sample)(Sample),6725中国科技论文在线(Sam2ple)(Sample),2,(6b).1.Marker;21Negativecontrol;31Sample;41Sample;5.Sample;61Sample5BDNAPCRFig.5ExtractedDNAfromSample,,,,..SmartView,DGGE,,.(2Band1).2616SrDNADGGEFig.616SrNDAseparatedbyDGGE776中国科技论文在线(Sample),3,.2(Sample)(6d),,.,.21416SrDNAABand17(6b)PCR,NCBI.216SrDNADGGETable2Thesequencesofseveral16SrDNADGGEfragmentsNCBIBand1Arcobactersp1A3b2(9815%)Arcobactersp.D1a1(9815%)Arcobactersp.B4b1(9815%)marinebacteriumMSC2(98.5%)Arcobactersp1KT0913(98.5%)10707837262121182459127749901BLASTQ3Band2unculturedeubacteriumWJGRT2117(9613%)unculturedepsilonproteobacterium(9010%)Sulfurospirillumsp.JPD21(8912%)unculturedproteobacterium(89.2%)107078381121247521054226607271BLASTQ3Band3,10707840252134422182563779494.BLASTQ3Band4unculturedbacteriumPHOS2HD43(9015%)Acinetobactersp.DVL00/879(9015%)Acinetobactersp.IrT2R5M22138(9015%)Acinetobacterhaemolyticus(9014%)Acinetobactersp.GOBB32C107(8919%)10707841052179162151282444648.BLASTQ3Band5Acinetobacterparvus(9715%)Acinetobactersp.Ni38(9619%)Acinetobactersp.An9(9619%)Acinetobacterjohnsonii(9619%)Acinetobactergenomosp.C1(9613%)10707841542139432149682629269.BLASTQ3Band6Acinetobactercalcoaceticus(9419%)bacteriumK87(9419%)Acinetobactersp.LBV326(9413%)Acinetobactersp.ADP1(9310%)Acinetobactersp.DSM586(9310%)10707841932140912122346313168.BLASTQ3Band7Sphaerotilussp.L12678121319(9614%)Leptothrixsp.L451011161718(9518%)Sphaerotilussp.IF14(9512%)betaproteobacteriumA0640(95.2%)Leptothrixsp.L14(9512%)10707842482182962158658938339.BLASTQ33(1),,.,,,,,..,,8725中国科技论文在线(2),..,,,,..(3)16SrDNA,,,.,,.,,,(6d6c).,,(6d).(4)16SrDNA,(2),Acineto2bacterArcobacter2.,,.,.