工业化废水处理反应器污泥总DNA提取方法

微生物学通报OCT20,2008,35(10):1659~1663Microbiology©2008byInstituteofMicrobiology,[e-mail]基金项目：(No.E2008000694,No.E200700063)*通讯作者：:[e-mail]收稿日期：2008-04-03;接受日期：2008-06-11生物实验室工业化废水处理反应器污泥总DNA提取方法张苏刘春*杨景亮郭建博李再兴(050018)摘要:根据工业化废水处理反应器污泥特性,对常规的溶菌酶-SDS-酚/氯仿环境样品总DNA提取方法进行改进,增强样品预处理,强化细胞裂解,提高杂质去除效率,获得了一种工业化污泥总DNA提取的通用方法,并采用该方法对石家庄若干实际运行的工业化厌氧、好氧反应器的污泥样品进行了总DNA提取研究。结果表明,该方法对所选污泥样品均有效,具有普适性。提取的污泥总DNA杂质含量少,纯度高,A260/A280在1.8左右;提取效率较高,总DNA产率都在0.7mg/g以上,最大产率可达0.85mg/g。所提取的污泥总DNA可以直接作为模板进行PCR反应,PCR产物直接进行变性梯度凝胶电泳(DGGE),能够得到较好的DGGE谱图,表明该方法提取的污泥总DNA样品可满足后续分析研究的要求。关键词:工业化废水处理反应器,厌氧/好氧污泥,总DNA,提取方法AMethodforTotalDNAExtractionofSludgeSamplesfromFull-scaleWastewaterTreatmentBioreactorsZHANGSuLIUChun*YANGJing-LiangGUOJian-BoLIZai-Xing(SchoolofEnvironmentalScienceandEngineering,HebeiUniversityofScienceandTechnology,Shijiazhuang050018)Abstract:Accordingtothecharacteristicsofsludgesamplesfromfull-scalewastewatertreatmentbioreac-tors,theessentialtotalDNAextractionmethodformostenvironmentalsamples,lysozyme-SDS-phenol/chloroformmethod,wasmodifiedtoimprovesamplepretreatment,intensifycelllysisandenhancetheeffi-ciencyofimpurityremoval.ObtainageneraltotalDNAextractionmethodforindustrialsludgesamples.SuchamethodwasappliedfortotalDNAextractionofsludgesamplesfromseveralrunningfull-scalean-aerobicoraerobicbioreactorsinShijiazhuang,China.Theresultsindicatedthatthemodifiedmethodwassuitableforallthesludgesamplesinthisstudy,showingthesatisfyinggenerality.TheextractedtotalDNAofallsludgesampleswerepure,withabout1.8ofA260/A280ratio.Themethodwasalsoefficient;withaveragetotalDNAyieldofover0.7mg/gandmaximumyieldof0.85mg/g.Moreover,alltheextractedto-talDNAsamplescouldserveastemplatesdirectlytoamplify16SrDNAbyPCR.ThePCRproductscouldbeseparatedwellbydenaturinggradientgelelectrophoresis(DGGE)andtheDGGEbandpatternswereclearenoughtobeusedforfurtheranalysis.AllthesefactsindicatedthatthetotalDNAextractionmethodprovidedinthisstudycouldmeettherequirementsofsludgesamplesresearch,fromfull-scalewastewatertreatmentbioreactors,usingmolecularbiologytechnologies.1660微生物学通报2008,Vol.35,No.10:Full-scalewastewatertreatmentbioreactor,Anaerobic/aerobicsludge,TotalDNA,Extractionmethod,,,DGGE、FISH、RAPD[1−5],DNADNA[6,7]。DNA,DNA,;;/[8,9]DNA[10−13],DNA,,,,,DNA,-SDS-/,DNA,,DNA,PCR-DGGEDNA1材料与方法1.1污泥样品1表1污泥样品性质Table1ThepropertiesofsludgesamplesSamplenumberWastewater-treating/Aerobic/anaerobic/Granular/flocBioreactorscale1234B12(VB12)5C(VC)671.2试剂和仪器(5mg/mL)6SDSK(10mg/mL)5mol/LNaClCTAB/NaCl(10mol/LCTAB,0.7mol/LNaCl)::(25:24:1,V/V/V):(24:1,V/V),(−20°C)SORVALLBiofugePrimoRUV2600DYY-6C,AppliedBiosystems2720PCRBio-RadGelDocXR1.3污泥总DNA提取方法(1)5mL,50mL3~4(2mm~3mm),:DNA1661~3min,,8000r/min5min,5mLTE(Tris100mmol/L,EDTA100mmol/L,pH8.0),2min,2(2),50mg/mL,5mg/mL,37°C225r/min,30min;1.5mL,1mL;6SDS200μL(1),,65°C,3h,20min;3h,,21.5mL,10mg/mLK6μL,55°C1h;100μL5mol/LNaCl,80μLCTAB/NaCl(10mol/LCTAB,0.7mol/LNaCl),65°C10min(3)DNA::(25:24:1,V/V/V),12000r/min5min,:(24:1,V/V),,12000r/min5min,,(4)DNA,−20°C12000r/min20min,,80100μLDNA,12000r/min5min,2,;100μL1μLRNaseA,37°C30min1.4总DNA样品的PCR扩增DNA,16SrRNAPCRBSF338(5′-ACTCCTACGGGAGGCAGCAG-3′)BSR534(5′-ATTACCGCGGCTGCTGGC-3′),GC5′-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-3′[14]PCR94°C3min;94°C1min,65°C45s,72°C45s,0.5°C,55°C,20;94°C1min;55°C45s,72°C45s,15;72°C8minPCR11.5PCR产物的变性梯度凝胶电泳(DGGE)分析DNAPCRBio-Rad(DGGE)8,30~60(1007mol/L40)60°C,60V,12hEBBio-RadQuantityOne2结果及讨论2.1工业化废水处理反应器污泥样品总DNA提取策略,DNA[15−17]DNADNA,,,-SDS-/,,DNA,,,;,TE,,[18]-SDS-/,SDS,20min1,SDS,SDSK,DNA;CTAB/NaCl,,DNA;,,,2.2污泥总DNA样品纯度及产率,DNA-,A260/A280,DNADNAA260/A280(1gDNA)2,DNAA260/A2801.8,DNA1662微生物学通报2008,Vol.35,No.10表2污泥样品总DNA样品纯度和产率Table2YieldandpurityofDNAproductsofdifferentsamplesSludgesamples1234567Purity(A260/A280)1.79331.81221.78231.80241.82751.80671.7945Yield(mg/g)0.70340.74530.69830.85050.82660.8050.8343,DNA,DNA0.7mg/g,0.85mg/g,DNADNA,,DNA,DNA,1,DNA23kb,RNA,DNA图1部分污泥样品总DNA琼脂糖电泳图Fig.1AgarosegelelectrophoresisoftotalDNAofsomesludgesamples注：样品No.1~No.5见表1Note:ThesamplesNo.1~No.5listinTable12.3污泥样品总DNA的PCR扩增,DNAPCR-DGGE[19]DNA,DNA,PCRPCR2,DNAPCR200bp,,DNAPCR[7],,2PCR,DNA,PCR,,图2部分总DNA样品PCR产物琼脂糖电泳图Fig.2AgarosegelelectrophoresisofPCRproductsofsometotalDNAsamples注：样品No.1~No.5见表1Note:ThesamplesNo.1~No.5listinTable1:DNA1663(No.2No.3)PCRDGGE3,PCR16SrDNADGGE,DGGE,PCR-DGGE,DNA,图3污泥样品2、3的DGGE图谱Fig.3DGGEprofilesofsludgesample2and3注：样品No.2和No.3见表1Note:ThesamplesNo.2和No.3inTable13结论(1),DNA,,(2)DNA,,A260/A2801.8;,,DNA0.7mg/g,0.85mg/g(3)DNAPCRDGGE,参考文献[1],,.FISHDGGE.,2006,27(11):2268−2272.[2],,,.PCR-DGGE.,2005,25(4):242−248.[3]CollinsG.Microbialcommunitystructureandmethano-genicactivityduringstart-uppsychrophilicanaerobicdi-gestertreatingsyntheticindustrialwastewaters.FEMSMicrobiologyEcology,2003,4(6):159−170.[4],,,..,2005,28(1):119−123.[5]BuzziniAP,SakamotoIK,VarescheMB,etal.EvaluationofthemicrobialdiversityinanUASBreactortreati