荧光原位杂交技术及其在微生物生态学中的应用-呼庆

第24卷第5期2004年5月生态学报ACTAECOLOGICASINICAVol.24,No.5May,2004荧光原位杂交技术及其在微生物生态学中的应用呼庆,齐鸿雁*,张洪勋(中国科学院生态环境研究中心环境生物技术研究室,北京100085)基金项目:国家“十五”科技攻关重点资助项目(2001BA903B)收稿日期:2003-10-21;修订日期:2004-01-10作者简介:呼庆(1977～),男,内蒙古呼和浩特市人,博士生,主要从事环境微生物分子生态学研究.*通讯作者Authorforcorrespondence。E-mail:qihy@mail.rcees.ac.cnFoundationitem:theNational“TenthFive-yearPlan”KeyTechnologiesR&DPrograme(No.2001BA903B)Receiveddate:2003-10-21;Accepteddate:2004-01-10Biography:HUQing,Ph.D.candidate,mainlyengagedinmolecularecologyofenvironmentalmicroorganism.摘要:综述了荧光原位杂交技术(fluorescenceinsituhybridizationFISH)在微生物生态学领域的各种应用,同时就其发展过程、原理及种类做了介绍。关键词:荧光原位杂交;微生物生态;16SrRNA;探针Fluorescenceinsituhybridization(FISH)anditsapplicationsinmicrobialecologyHUQing,QIHong-Yan*,ZHANGHong-Xun(EnvironmentalBiotechnologyLab.,ResearchCenterforEco-environmentalSciences,ChineseAcademyofSciences,Beijing100085,China).ActaEcologicaSinica,2004,24(5):1048～1054.Abstract:Duringrecentyears,moleculartechniquessuchasPCRanddenaturinggradientgelelectrophoresis(DGGE)orDNAsequencinghaverevolutionizedallfieldsofmicrobiology,andsensitivedetectionandexactidentificationofbacteriaarepossible.Fluorescenceinsituhybridization(FISH)using16SrRNAprobesdoesnotrelyonPCRamplification,andassuchprovidesausefulcomplementarytechniquetoDGGEfortheanalysisoforganisms.BecauseofFISHallowingnucleicacidsequencestobeexaminedinsidecellswithoutalteringthecell'smorphologyortheintegrityofitsvariouscompartmentsandprovidinginformationaboutnumber,spatialdistributionandcellularenvironment,ithasbecomeapowerfultoolforphylogenetic,ecologic,diagnosticandenvironmentalstudiesinmicrobiology.Fluorescenceinsituhybridizationcandetectnucleicacidsequencesbyafluorescentlylabeledprobethathybridizesspecificallytoitscomplementarytargetsequencewithintheintactcell.ItsgeneralprocedureinFISHanalysisofmicroorganismsisasfollows:(1)fixationofthespecimen;(2)preparationofthesample,possiblyincludingspecificpretreatmentsteps;(3)hybridizationwiththerespectiveprobesfordetectingtherespectivetargetsequences;(4)washin