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26120072SICHUANENVIRONMENTVol126,No11February2007:2006207210:(NCET20520387);(20050247016):(1982-),,,2004,,(,200092):PCR2DGGE,AN/AODNA,PCR(nestedPCR)PCR,2Proteobacteria2Proteobacteria16SrDNADGGE(),NCBI(),2Proteobacteria2Proteobacteria,,PCR2DGGE:;;;;:X172:A:100123644(2007)0120009205PCR2DGGEMethodsfortheAnalysisofCommunityStructureofPhosphorusAccumulatingOrganismsDAIRui,LIUHong2bo,XIASi2qing(StateKeyLaboratoryofPollutionControl&Reuse,TongjiUniversity,Shanghai200092,China)Abstract:TotalDNAwasdirectlyextractedfromactivatedsludgeofsixreactorsofAN/AOprocess.NestedPCRandDGGEwereusedtoamplifyandseparate16SrDNAof2Proteobacteriaand2Proteobacteria.Thesequenceofseveral16SrDNADGGEfragmentswasdeterminedthroughcomparisoninGenBank(NCBI).ItwasconfirmedthatthespeciesofProteobacteriawereabundantintheEnhancedBiologicalPhosphorusRemoval(EBPR)Processes.TheresultsshowedthatthePCR2DGGEtechnologycombinedwithsequencesdeterminationisanefficientmethodfortheanalysisofcommunitystructureofphosphorusaccumulatingorganismsinwastewatertreatmentprocess.Keywords:PCR;DGGE;biologicalphosphorusremoval;activatedsludge;Proteobacteria(EBPR)()(),[1],,,[2]PCR-DGGE,,,,(Proteobacteria)[3],PCR2DGGE(polymerasechainreaction2denaturtinggradientgelelectrophoresis)2Proteobacteria2Pro2teobacteria,DNA,2Proteobacteria2Proteobacteria16SrDNA,PCRDGGE,PCR(nestedPCR)[4]2Proteobacteria2ProteobacteriaPCR;PCR,16SPCR;DGGE,PCRPCR,PCR,,DGGE1111AN/AO(1),111,[5]CODCr200mg/L,TN4060mg/L,TP510mg/L,CODCr49mg/L,TN1412mg/L,017mg/L,517%(gP/gMLSS),1AN/AOFig11SchematicdiagramoftheparallelAN/AOprocess50mL,30min,,1mL115mL10min(9000r/min,25),100mgDNA121212SampleSam2pleSampleSampleSampleSample(28)11211211DNADNADNADNA015%(015g,015TBE100mL),Marker()-Hinddigest11212PCR2ProteobacteriaPCR[6]:P1:52CCGCATACGCCCTACGGGGGAAAGATTTAT23,P2:52CGGTGTGTACAAGGCCCGGGAACG23,[5]:95,10min;95,60s,56,60s,72,120s,35cycles;72,10min:40L,10PCR5L,4dNTP1L,10mmol/L1L,Taq1,DNA1L2ProteobacteriaPCR[7]:P1:52CCTACGGGAGGCAGCAG23,P2:52CCCCGTCAATTCCTTTGAGTTT23,[6]:94,2min;94,60s,53,60s,72,60s,30cy2cles;72,2min:40L,10PCR5L,4dNTP1L,10mmol/L1L,Taq1,DNA1L2Proteobacteria2Proteobacteria16SrDNAV3[8](P1:52ATTACCGCGGCTGCTGG23,P2:CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCCCCTACGGGAGGCAGCAG23),GCDGGE[8],[9]:94,10min;94,45s,60,45s,72,90s,30cycles;72,10min:80L,10PCR10L,4dNTP2L,10mmol/L2L,Taq2,PCR2LPCR1%,Marker()DNAMarkerDL200011213DGGEPCRDGGE,:2Proteobacteria:50%65%,2Proteobacteria:30%70%;01261TAE[10],150V,60,715h,DNA,015mL,30L,PCR,2211DNA26DNA,markerSmartView,DNA23kb,DNADNA,,DNA2DNAFig12AgarosegelelectrophoresisofDNAextraction212DNAPCRDNA2Proteobacteria2ProteobacteriaPCR,2Proteobacteria1000bp(3),2Proteobacteriai700bp(4),,,PCR32ProteobacteriaPCRFig13AgarosegelelectrophoresisoffirstroundPCRamplifiedDNAof6samples42ProteobacteriaPCRFig14AgarosegelelectrophoresisoffirstroundPCRamplifiedDNAof6samplesPCR16SrDNAPCR,,DNA220240bp(56),16SrDNAV3,,PCR52ProteobacteriaPCRFig15AgarosegelelectrophoresisofsecondroundPCRamplifiedDNAof6sample62ProteobacteriaPCRFig16AgarosegelelectrophoresisofsecondroundPCRamplifiedDNAof6sample21316SrDNADGGEPCR,78,2Proteobacteria2ProteobacteriaDGGE2Proteobacteria2Proteobacteria,111:17(Band1Band7)72Proteobacteria16SrDNADGGEFig1716SrDNAseparatedbyDGGE82Proteobacteria16SrDNADGGEFig1816SrDNAseparatedbyDGGEAN/AO6,2Proteobacteria2Proteobacteria2Proteobacteria2Proteobacteria;,,2Proteobacteria2ProteobacteriaAN/AO2Pro2teobacteria2Proteobacteria,21416SrDNABand1Band7PCR,NCBI,,16SrDNA,AN/AO2Proteobacteria2ProteobacteriaDevosiasp1Paracoccussp1Bdellovibriosp1,16SrDNADGGETab1Thesequenceofseveral16SrDNADGGEfragmentNCBIBand1Devosiasp.PIC2C2295%Unculturedalphaproteobacterium95%11472574322296762152079535703.BLASTQ1Band2Unculturedsludgebacterium97%Unculturedfreshwaterbacterium97%1147261976222196245234316334.BLASTQ4Band3UnculturedRhodobacteraceaebacterium98%Paracoccussp.L7K597%1147263339210552251451698221.BLASTQ4Band4UnculturedsludgebacteriumA10100%UnculturedsludgebacteriumA2698%1147264073222943227439731208.BLASTQ4Band5Bdellovibriosp.99%Unculturedbacterium98%11472652202287092194999633185.BLASTQ1Band6UnculturedCytophagasp.98%UnculturedBacteroidetesbacterium97%UnculturedbacteriumCYB277100%1147269399216600230616484735.BLASTQ4Band7Bdellovibriosp.L100%Bdellovibriosp.Ec13100%Bdellovibriosp.CHI100%11472664902147412206762013579.BLASTQ4,Bdellovibriosp.;De2vosiasp.;Paracoccussp.3DNA,;PCR2DGGE,;PCRPCRDGGE,2126AN/AO2Proteobacteria2Pro2teobacteria16SrDNA,2Proteobacte2ria2ProteobacteriaDGGE,,2Pro2teobacteria2Proteobacteria,Devosiasp.Paracoccussp.Bdell2ovibriosp.,,Devosiasp.Paracoccussp.Bdellovibriosp.,,:[1],.[J].,2004,23(6):41244.[2]SatoshiTsuneda,RyukiMiyauchi,TakashiOhno,etal.Character2izationofDenitrifyingPolyphosphate-AccumulatingOrganismsinActivatedSludgeBasedonNitriteReductaseGene[J].Journalofbioscienceandbioengineering,2005,99(4):4032407.[3]RobertJ.Seviour,TakashiMino,MotoharuOnuki.Themicrobiol2ogyofbiologicalphosphorusremovalinactivatedsludgesystem[J].FEMSMicrobiologyReviews,2003,27:992127.[4]ValeriaMaiadeOliveira,GilsonPauloManfio,HeitorLuizdaCostaCo