真菌检测鉴定通用引物FungalPrimers

ITS1:5’-CCGTAGGTGAACCTGCGG-3’ITS4:5’-TCCTCCGCTTATTGATATGC-3’Tm55℃NS17:CATGTCTAAGTTTAAGCAANS3:GCAAGTCTGGTGCCAGCAGCCNS4:CTTCCGTCAATTCCTTTAAGNS22:AATTAAGCAGACAAATCACTNS24:AAACCTTgTTACgACTTTTALR0R:5’-GTACCCGCTGAACTTAAGC-3’LR3:5’-CCGTGTTTCAAGACGGGLR3R:5’-GTCTTGAAACACGGACC(complementarytoRLR3R:GGTCCGTGTTTCAAGAC)LR5:5’-TTAAAAAGCTCGTAGTTGAAC-3’LR7:5’-TACTACCACCAAGATCTLR12:5’-GACTTAGAGGCGTTCAGLr0R/LR5:Tm50-52℃NL1:5’-GCATATCAATAAGCGGAGGAAAAGNL1:5′-TGCGTTGATTACGTCCCTGC(alsocalledV9:TGCGTTGATTACGTCCCTGC)NL1:5’-TGCTGGAGCCATGGATC-3NL2:5’-CTCTCTTTTCAAAGTTCTTTTCATCTNL2:5’-AACGGCTTCGACAACAGC-3NL2:5’-CTTGTTCGCTATCGGTCTC(alsoNL2A:5′-CTTGTTCGCTATCGGTCTC)NL2:5’-TACTTGTTCGCTATCGGTCT-3'NL3:5’-GAGACCGATAGCGAACAAG(alsoNL3A:5’-GAGACCGATAGCGAACAAG)NL3:5’-AGACCGATAGCGAACAAGTANL3:5’-GCTGTTGTCGAAGCCGTT-3NL4:5’-GGTCCGTGTTTCAAGACGG（similartoRLR3R：5′-GGTCCGTGTTTCAAGAC)NL4:5’-TAGATACATGGCGCAGTC-3ConservedprimersequencesforPCRamplificationandsequencingfromnuclearribosomalRNA(HYPERLINK"http://www.biology.duke.edu/fungi/mycolab/primers.htm"http://www.biology.duke.edu/fungi/mycolab/primers.htm)Vilgalyslab,DukeUniversityOvertheyears,ourlabhascompiledausefullistofconservedprimersequencesusefulforamplificationandsequencingofnuclearrDNAfrommostmajorgroupsoffungi(primarilyEumycota),aswellasothereukaryotes.AlloftheseprimerswereidentifiedandtestedbyourownlabbasedonconsensusbetweenthepublishedlargeandsmallsubunitRNAsequencesfromfungi,plantsandothereukaryotes;sourcesofotherusefulprimersequencesfrompublishedliteraturearealsoindicated.Together,theseprimersspanmostofthenuclearrDNAcodingregion(seefigures),permittingamplificationofanydesiredregion.Standardsymbolsareusedforthefourprimarynucleotides;variablepositionsareindicatedasfollows:P=A,G/Q=C,T/R=A,T/V=A,C/W=G,T.Primersendingwith"R"representthecodingstrand(sameasRNA).Allotherprimersarecomplementarytothecodingstrand.Thisinformationisprovidedfreelyandmaybepassedontoanyonewhowantstouseit.Thenuclear-encodedribosomalRNAgenes(rDNA)offungiexistasamultiple-copygenefamilycomprisedofhighlysimilarDNAsequences(typicallyfrom8-12kbeach)arrangedinahead-to-toemanner.Eachrepeatunithascodingregionsforonemajortranscript(containingtheprimaryrRNAsforasingleribosome),punctuatedbyoneormoreintergenicspacer(IGS)regions.Insomegroups(mostlybasidiomcyetesandsomeascomycetousyeasts),eachrepeatalsohasaseparatelytranscribedcodingregionfor5SRNAwhosepositionanddirectionoftranscriptionmayvayamonggroups.SeveralrestrictionsitesforEcoRIandBglIIareconservedintherDNAoffungi.Nearlyallbasidiomyceteswe'vestudiedshareanEcoRIsitewithinthe5.8SRNAgenealongwithaBglIIsitehalfwayintotheLSURNAsequence.Primers5.8SRandLR7includetheserestrictionsites,whichmakesthemconvenientforcloning.Forthosewhoaren'tfamiliarwithrDNAandfungalsystematics,severalexcellentreviewsareavailableonfungi(Hibbett,1992)andgenerallyforeukaryotes(HillisandDixon,1991).SeeGerbi(1986)forageneralintroductiontothemolecularbiologyandevolutionofrDNAinothereukaryotes.AnotherusefulsourceofprimerinformationmaybefoundinGargas&Depriest(1996)andattheTomBrunslabwebsiteHYPERLINK"http://mendel.berkeley.edu/boletus/boletus.html"http://mendel.berkeley.edu/boletus/boletus.html.SmallsubunitRNA(SR)primers:PrimernameSequence(5'-->3')PositionwithinS.cereviseae17SRNABMB-'A'GRATTACCGCGGCWGCTG580-558BMB-'B'CCGTCAATTCVTTTPAGTTT1146-1127BMB-'C'ACGGGCGGTGTGTPC1638-1624BMB-BRCTTAAAGGAATTGACGGAA1130-1148BMB-CRGTACACACCGCCCGTCG1624-1640SR1RTACCTGGTTGATQCTGCCAGT1-21SR1ATTACCGCGGCTGCT578-564SR2CGGCCATGCACCACC1277-1263SR3GAAAGTTGATAGGGCT318-302SR4AAACCAACAAAATAGAA838-820SR5GTGCCCTTCCGTCAATT1146-1130SR6TGTTACGACTTTTACTT1760-1744SR6RAAGWAAAAGTCGTAACAAGG1744-1763SR7GTTCAACTACGAGCTTTTTAA617-637SR7RAGTTAAAAAGCTCGTAGTTG637-617SR8RGAACCAGGACTTTTACCTT732-749SR9RQAGAGGTGAAATTCT896-910SR10RTTTGACTCAACACGGG1181-1196NS1GTAGTCATATGCTTGTCTCNS2GGCTGCTGGCACCAGACTTGCNS3GCAAGTCTGGTGCCAGCAGCCNS4CTTCCGTCAATTCCTTTAAG(similartoBMB-B)NS5AACTTAAAGGAATTGACGGAAG(issimilartoBMB-BR)NS6GCATCACAGACCTGTTATTGCCTCNS7GAGGCAATAACAGGTCTGTGATGCNS8TCCGCAGGTTCACCTACGGABMB="universal"SSUprimersdevelopedbyLaneetal.,1985SR=primersdevelopedbyVilgalyslabNS=primersdescribedbyWhiteetal.,1990LargesubunitRNA(25-28S)primersequencesNote:mostmolecularsystematicsstudiesonlyutilizethefirst600-900basesfromtheLSUgene,whichincludesthreedivergentdomains(D1,D2,D3)thatareamongthemostvariableregionswithintheentiregene(muchoftheLSUisinvariantevenacrosswidelydivergenttaxa).MostofthedatainourAgaricalesLSUdatabaseconsistsofthefirst900basesfromtheLSUgene(wetypicallyamplifyusingprimers5.8SR+LR7,followedbysequencingusingprimersLR5,LR16,LR0R,andLR3R).PrimernameSequence(5'-->3')PositionwithinS.cereviseaerRNAcomments5.8SCGCTGCGTTCTTCATCG51-35(5.8SRNA)containsEcoRIsite5.8SRTCGATGAAGAACGCAGCG34-51(5.8SRNA)containsEcoRIsiteLR0RACCCGCTGAACTTAAGC26-42LR1GGTTGGTTTCTTTTCCT73-57LR2TTTTCAAAGTTCTTTTC385-370LR2RAAGAACTTTGAAAAGAG374-389LR3CCGTGTTTCAAGACGGG651-635LR3RGTCTTGAAACACGGACC638-654LR4ACCAGAGTTTCCTCTGG854-838LR5TCCTGAGGGAAACTTCG964-948LR6CGCCAGTTCTGCTTACC1141-1125LR7TACTACCACCAAGATCT1448-1432containsBglIIsiteLR7RGCAGATCTTGGTGGTAG1430-1446containsBglIIsiteLR8CACCTTGGAGACCTGCT1861-1845LR8RAGCAGGTCTCCAAGGTG1845-1861LR9AGAGCACTGGGCAGAAA2204-2188LR10AGTCAAGCTCAACAGGG2420-2404LR10RGACCCTGTTGAGCTTGA2402-2418LR11GCCAGTTATCCCTGTGGTAA2821-2802LR12GACTTAGAGGCGTTCAG3124-3106LR12RCTGAACGCCTCTAAGTCAGAA3106-3126LR13CGTAACAACAAGGCTACT3357-3340LR14AGCCAAACTCCCCACCTG2616-2599LR15TAAATTACAACTCGGAC154-138LR16TTCCACCCAAACACTCG1081-1065LR17RTAACCTATTCTCAAACTT1033-1050LR20RGTGAGACAGGTTAGTTTTACCCT2959-2982LR21ACTTCAAGCGTTTCCCTTT424-393LR22CCTCACGGTACTTGTTCGCT364-344Internaltranscribedspacer(ITS)regionprimersTheITSregionisnowperhapsthemostwidelysequencedDNAregioninfungi.Ithastypicallybeenmostusefulformolecularsystematicsatthespecieslevel,andevenwithinspecies(e.g.,toidentifygeographicraces).BecauseofitshigherdegreeofvariationthanothergenicregionsofrDNA(SSUandLSU),variationamongindividualrDNArepeatscansometimesbeobservedwithinboththeITSandIGSregions.InadditiontothestandardITS1+ITS4primersusedbymostlabs,everaltaxon-specificprimershavebeendescribedthatallowselectiveamplificationoffungalsequences(e.g.,seeGardes&Bruns1993paperdescribingamplificationofbasidiomyceteITSsequencesfrommycorrhizasamples).primernamesequence(5'->3')commentsreferenceITS1TCCGTAGGTGAACCTGCGGWhiteetal,1990ITS2GCTGCGTTCTTCATCGATGC(issimilarto5.8Sbelow)Whiteetal,1990ITS3GCATCGATGAAGAACGCAGC(issimilarto5.8SRbelow)Whiteetal,1990ITS4TCCTCCGCTTATTGATATGCWhiteetal,1990ITS5GGAAGTAAAAGTCGTAACAAGG(issimilartoSR6R)Whiteetal,1990ITS1-FCTTGGTCATTTAGAGGAAGTAAGardes&Bruns,1993ITS4-BCAGGAGACTTGTACACGGTCCAGGardes&Bruns,19935.8SCGCTGCGTTCTTCATCGVilgalyslab5.8SRTCGATGAAGAACGCAGCGVilgalyslabSR6RAAGWAAAAGTCGTAACAAGGVilgalyslabIntergenicspacer(IGS)primers(including5SRNAprimersequencesforbasidiomycetefungi)ThegreatestamountsequencevariationinrDNAexistswithintheIGSregion(sometimesalsoknownasthenon-transcribedspacerorNTSregion).ThesizeoftheIGSregionmayvaryfrom2kbupwards.Itisnotunusualtofindhypervariabilityforthisregion(necessitatingcloningofindividualrepeathaplotypes).Severalpatternsoforganizationcanbefoundindifferentgroupsoffungi:MostfilamentousascomyceteshaveasingleuninterruptedIGSregion(betweentheendoftheLSUandstartofthenextSSUsequence),whichmayvaryinlengthfrom2-5kbormore.AmplificationoftheentireIGSregionrequiresusingprimersanchoredinthe3'endoftheLSUgene(e.g.,LR12R)and5'endoftheSSURNAgene(e.g.,invSR1R).Inmanyascomycetousyeastsandnearlyallbasidiomycetes,theIGSalsocontainsasinglecodingregionforthe5SRNAgene,whichdividestheIGSintotwosmallerregionsthatmaybemoreeasilyamplifiedusing.Dependingontheorientationandpositionofthe5SRNAgene,thePCRmaybeusedtosequentiallyamplifyeitheraportionoftheintergenicspacerregion(IGS)beyondthelargesubunitRNAcodingregion.primersequence(5'->3')commentsreferenceLR12RGAACGCCTCTAAGTCAGAATCClocatedwithintheLSURNA(seeabove)VilgalyslabinvSR1RACTGGCAGAATCAACCAGGTAlocatedwithintheSSURNA(positions21-1)Vilgalyslab5SRNAATCAGACGGGATGCGGT(complementaryto5SRNApositions46-26)Vilgalyslab5SRNARACQGCATCCCGTCTGAT(5SRNApositions26-46)VilgalyslabREFERENCESBruns,T.D.,R.Vilgalys,S.M.Barns,D.Gonzalez,D.S.Hibbett,D.J.Lane,L.Simon,S.Stickel,T.M.Szaro,W.G.Weisburg,andM.L.Sogin.1992.Evolutionaryrelationshipswithinthefungi:analysesofnuclearsmallsubunitrRNAsequences.Molec.Phylog.Evol.1:231-241.Bruns,T.D.,T.J.White,andJ.W.Taylor.1991.Fungalmolecularsystematics.Ann.Rev.Ecol.Syst.22:525-564.DePriest,P.T.,andM.D.Been.1992.NumerousgroupIintronswithvariabledistributionsintheribosomalDNAofalichenfungus.J.Mol.Biol.228:315-321.Elwood,H.J.,G.J.Olsen,andM.L.Sogin.1985.ThesmallsubunitribosomalRNAgenesequencesfromthehypotrichousciliatesOxytrichanovaandStylonychiapustula.Mol.Biol.Evol.2:399-410.Gardes,M.,andT.D.Bruns.1993.ITSprimerswithenhancedspecificityforbasidiomycetes-applicationtotheidentificationofmycorrhizaeandrusts.Mol.Ecol.2:113-118.Gargas,A.,andP.T.DePriest.1996.AnomenclatureforfungalPCRprimerswithexamplesfromintron-containingSSUrDNA.Mycologia88:745-748Gargas,A.,andJ.W.Taylor.1992.Polymerasechainreaction(PCR)primersforamplifyingandsequencing18SrDNAfromlichenizedfungi.Mycologia84:589-592.Gerbi,S.A.1986.Chapter7-EvolutionofribosomalDNA.Pp.419-517In:Molecularevolution,ed.McIntyre,R.Hibbett,D.S.1991.PhylogeneticrelationshipsoftheBasidiomycetegenusLentinus:evidencefromribosomalRNAandmorphology.Ph.D.Thesis,DukeUniversity,1991.Hibbett,D.S.1992.RibosomalRNAandfungalsystematics.Trans.Mycol.Soc.Jpn.33:533-556.Hibbett,D.S.,andR.Vilgalys.1991.EvolutionaryrelationshipsofLentinustothePolyporaceae:evidencefromrestrictionanalysisofenzymaticallyamplifiedribosomalDNA.Mycologia83:425-439.Hibbett,D.S.,andR.Vilgalys.1993.PhylogeneticrelationshipsoftheBasidiomycetegenusLentinusinferredfrommolecularandmorphologicalcharacters.Syst.Bot.18:409-433.Hillis,D.M.,andM.T.Dixon.1991.RibosomalDNA:molecularevoluitonandphylogeneticinference.Quart.Rev.Biol.66:411-453.Hopple,J.S.,Jr.,andR.Vilgalys.1994.PhylogeneticrelationshipamongcoprinoidtaxaandalliesbasedondatafromrestrictionsitemappingofnuclearrDNA.Mycologia86:96-107.Lane,D.J.,B.Pace,G.J.Olsen,D.A.Stahl,M.L.Sogin,andN.R.Pace.1985.Rapiddeterminationof16SribosomalRNAsequencesforphylogeneticanalyses.Proc.Natl.Acad.Sci.,U.S.A.82:6955-6959.Vilgalys,R.,andD.Gonzalez.1990.OrganizationofribosomalDNAinthebasidiomyceteThanatephoruspraticola.Curr.Genet.18:277-280.Vilgalys,R.,J.S.Hopple,Jr.,andD.S.Hibbett.1994.Phylogeneticimplicationsofgenericconceptsinfungaltaxonomy:Theimpactofmolecularsystematicstudies.MycologicaHelvetica6:73-91.White,T.J.,T.Bruns,S.Lee,andJ.W.Taylor.1990.AmplificationanddirectsequencingoffungalribosomalRNAgenesforphylogenetics.Pp.315-322In:PCRProtocols:AGuidetoMethodsandApplications,eds.Innis,M.A.,D.H.Gelfand,J.J.Sninsky,andT.J.White.AcademicPress,Inc.,NewYork.©W°