厌氧活性污泥微生物群落的SSCP分析条件优化

厌氧活性污泥微生物群落的SSCP分析条件优化摘要针对厌氧活性污泥微生物群落结构复杂,群落中相似基因序列较多等特点,对影响单链构像多态性(SSCP)图谱分辨率的电泳温度、甘油、交联度和影响聚合酶链式反应(PCR扩增)的BSA等因素进行了实验研究。结果表明,选择细菌16SrDNAV3区(约200bp)PCR扩增片段,在凝胶浓度为12%、丙烯酰胺与双丙烯酰胺质量比为49:1、无甘油等条件下,恒压260V,4℃电泳17h,可获得较为理想的SSCP图谱;BSA的使用,有助于降低样品中残留腐殖酸和棕黄酸等杂质对PCR扩增的抑制作用,提高SSCP图谱的分辨率和可读性,为厌氧活性污泥细菌群落结构解析提供了有效手段。关键词分子生物学;聚合酶链式反应;单链构像多态性;厌氧活性污泥;群落结构;牛血清白蛋白中图分类号X703文献标识码A文章编号1000-7857(2008)02-0028-05鲍立新,李建政,赵焱,郑国臣哈尔滨工业大学市政环境工程学院,哈尔滨150090OptimizationofSingle-StrandConformationPolymorphismTechnologyforAnalyzingMicrobialCommunityofAnaerobicActivatedSludgeBAOLixin,LIJianzheng,ZHAOYan,ZHENGGuochenSchoolofMunicipalandEnvironmentalEngineering,HarbinInstituteofTechnology,Harbin150090,ChinaAbstractThemicrobialcommunitystructureofanaerobicactivatedsludgeisverycomplexwithmanysimilarmicrobegenesequences.TheSingle-StrandConformationPolymorphism(SSCP)technologyisproposedinthisrespect.InordertoobtainanSSCPprofilewithaperfectresolutiondetail,thefactors,includingelectrophoresistemperature,glycerinandgelstrengththatmayinfluencetheresolutionoftheSSCPprofile,andBovineSerumAlbumin(BSA)thatmayinfluencetheamplificationofthePolymerase-Chain-Reaction(PCR),areinvestigatedinthispaper.TheresultsshowthatSSCPprofilesofanaerobicactivatedsludgemicrobialcommunity,,amplifiedfromtheV3regionof16SrDNAgenes(about200bp),areperfectinresolutionsensitivityunderthefollowingconditions:thegelconcentrationof12%,theratioofacrylamide(AM)andN,N′-Methylenebisacrylamide(DMAM)of49∶1,withoutglycerin,electrophoresisat4℃and260Vfor17hours.TheuseofBSAinpretesttreatmentofanaerobicactivatedsludgecouldweakentheinhibitingeffectofhumicacidandfulvicacidonPCRamplificationandimprovetheresolutionsensitivityofSSCPprofiles.Theoptimizedtechniquecanbeusedformicrobialcommunityanalysisofanaerobicactivatedsludge.Keywordsmolecularbiology;polymerasechainreaction;single-strandconformationpolymorphism;anaerobicactivatedsludge;communitystructure;bovineserumalbumin0引言厌氧生物处理技术被视为高浓度有机废水处理的最为经济有效的方法,但仍然存在处理负荷低,运行控制难度大等不足。为了在厌氧生物处理效能提高和运行控制技术方面取得突破,对参与厌氧消化过程