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1、第24卷第5期生态学报Vol.24,No.52004年5月ACTAECOLOGICASINICAMay,2004荧光原位杂交技术及其在微生物生态学中的应用3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)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)
2、anditsapplicationsinmicrobialecology3HUQing,QIHong2Yan,ZHANGHong2Xun(EnvironmentalBiotechnologyLab.,ResearchCenterforEco2environmentalSciences,ChineseAcademyofSciences,Beijing100085,China).ActaEcologicaSinica,2004,24(5):1048～1054.Abstract:Duringrecentyears,moleculartech
3、niquessuchasPCRanddenaturinggradientgelelectrophoresis(DGGE)orDNAsequencinghaverevolutionizedallfieldsofmicrobiology,andsensitivedetectionandexactidentificationofbacteriaarepossible.Fluorescenceinsituhybridization(FISH)using16SrRNAprobesdoesnotrelyonPCRamplification,and
4、assuchprovidesausefulcomplementarytechniquetoDGGEfortheanalysisoforganisms.BecauseofFISHallowingnucleicacidsequencestobeexaminedinsidecellswithoutalteringthecell'smorphologyortheintegrityofitsvariouscompartmentsandprovidinginformationaboutnumber,spatialdistributionandce
5、llularenvironment,ithasbecomeapowerfultoolforphylogenetic,ecologic,diagnosticandenvironmentalstudiesinmicrobiology.Fluorescenceinsituhybridizationcandetectnucleicacidsequencesbyafluorescentlylabeledprobethathybridizesspecificallytoitscomplementarytargetsequencewithinthe
6、intactcell.ItsgeneralprocedureinFISHanalysisofmicroorganismsisasfollows:(1)fixationofthespecimen;(2)preparationofthesample,possiblyincludingspecificpretreatmentsteps;(3)hybridizationwiththerespectiveprobesfordetectingtherespectivetargetsequences;(4)washingstepstoremoveu
7、nboundprobes;(5)mounting,visualizationanddocumentationofresults.BecauseFISHgivesadetailedpictureofthemicroenviromentswithoutanyselectivepurificationoramplificationsteps,thereisalargescopeofFISHapplications.Ithasbeenextensivelyusedinthefieldofenvironmentalmicroorganismsd
8、iversitysuchastheinvestigationofmicrobialcommunitiesofaquatichabitatsandsoilhabitats.Mostobligatemicrobialsymb
