線蟲鑒定通用引物

TheprimerpairsusedwereD2A(5'-acaagtaccgtgagggaaagttg-3‘)andD2B(5'-aatccgtgtttcaagacggg-3'),D3A(5'-gacccgtcttgaaacacgga-3')andD3B(5'-tcggaaggaaccagctacta-3')(Nunn,1992),andmtA(5'-ggcggatcctacatcgatgttgtat-3')andmtB(5'-ggcggatccwkttcctctcgtact-3').TheseprimersselectivelyamplifymetazoanribosomalDNAs(rDNA)anddonotamplifycommoncontaminantssuchasbacteria,fungi,orplantmaterial.CourtrightEM,WallDH,VirginiaRA,etal.NuclearandmitochondrialDNAsequeneediversityintheAntarcticnematodeScottnemalindsayae.JournalofNematology,2000,32(2):143.DNAextractionandpolymerasechainreaction(PCR)assayswereperformedaccordingtoSubbotinetal.(2000).TheITS1-5.8S-ITS2andtheD2-D3of28SofrDNAwereamplifiedusingthefollowingprimersets:5367(5'-ttgattacgtccctgcccttt-3')andF195(5'-tcctccgctaaatgatatg-3');andD2A(5'-acaagtaccgtgagggaaagttg-3')andD3B(5'-tcggaaggaaccagctacta-3')asdescribedbySchmitzetal.(1998)andDeLeyetal.(1999),respectively.CastilloP,VovlasN,SubbotinS,etal.Anewroot-knotnematode,Meloidogynebaetican.sp.(Nematoda:Heteroderidae),parasitizingwildoliveinSouthernSpain.Phytopathology,2003,93(9):1093-1102.D3A(5'-gacccgtcttgaaacacgga-3')andD3B(5'-tcggaaggaaccagctacta-3').Al-BannaL,PloegAT,WilliamsonVM,etal.DiscriminationofsixPratylenchusspeciesusingPCRandspecies-specificprimers.Journalofnematology,2004,36(2):142.Thenuclearribosomalinternaltranscribedspacer(ITS1)segmentwasamplifiedwiththeprimersrDNA25'-ttgattacgtccctgcccttt-3'(Vrainetal.1992)andrDNA1.58S5'-acgagccgagtgatccaccg-3'(Cherryetal.1997).TheribosomalLSUD2-D3expansionsegmentwasamplifiedwithprimersD2A5'-acaagtaccgtgagggaaagttg-3'andD3B5'-tcggaaggaaccagctacta-3'(Courtrightetal.,2000)aspreviouslydescribed(Al-Bannaetal.,1997).SkantarAM,CartaLK.Multipledisplacementamplification(MDA)oftotalgenomicDNAfromMeloidogynespp.andcomparisontocrudeDNAextractsinPCRofITS1,28SD2-D3rDNAandHsp90.Nematology,2005,7(2):285-294.SeveralnematodespecimensofeachsampleweretransferredtoanEppendorftubecontaining16ulddH2O,2ul10XPCRbufferand2ulproteinaseK(600ug/ml)(Promega,Benelux,TheNetherlands)andcrushedfor2minwithaVibroMixermicrohomogeniser(ZCrich,Switzerland).Thetubeswereincubatedat65°C(1h)andthenat95°C(15min).DetailedprotocolsforPCR,cloningandautomatedsequencingaredescribedbyTanhaMaafietal.(2003).TheforwardD2A(5'-acaagtaccgtgagggaaagttg-3')andreverseD3B(5'-tcggaaggaaccagctacta-3')primerswereusedforamplificationandsequencingofthefragmentofthe28SrRNAgene.SubbotinSA,VovlasN,CrozzoliR,etal.PhylogenyofCriconematinaSiddiqi,1980(Nematoda:Tylenchida)basedonmorphologyandD2-D3expansionsegmentsofthe28S-rRNAgenesequenceswithapplicationofasecondarystructuremodel.Nematology,2005,7(6):927-944.ThedifferentregionsofrDNAwereamplifiedasdescribedbyCastilloetal.(2003)andTiganoetal.(2005)usingthefollowingprimersets:MelF(5'-tacggactgagataatggt-3')andMelR(5'-ggttcaagccactgcga-3')forthe18S,5367(5'-ttgattacgtccctgcccttt-3')andF195(5'-tcctccgctaaatgatatg-3')fortheITS15?8S-ITS2,D2A(5'-acaagtaccgtgagggaaagttg-3')andD3B(5'-tcggaaggaaccagctacta-3')fortheD2-D3regionof28S.RiusJEP,VovlasN,TroccoliA,etal.Anewroot-knotnematodeparasitizingsearocketfromSpanishMediterraneancoastaldunes:Meloidogynedunensisn.sp.(Nematoda:Meloidogynidae).Journalofnematology,2007,39(2):190.Cloning,characterisationandheterologousexpressionofanastacinmetalloprotease,Sc-AST,fromtheentomoparasiticnematodeSteinernemacarpocapsaeYJing,DToubarro,YHao,NSimoes-Molecularandbiochemical 2010-Elsevier...TotalRNAfromdifferentnematodestagesandfromnematodesinducedfor0,6,12,24,36,48and72hwas...Primersfor18SrRNAwere18SF(5'-GCTAATCGGAAACGAAAGTC-3')and18SR(5'-CATCCACCGAATCAAGAAAG-3').PrimersforSc-ASTwereASTF1(5...5.8SF194/F195: Tm55°CTW81/AB28:18S:Me1f/Me1r:Tm50C18SF/18SR:28S:D2A/D3B: Tm55CmtDNAC2F3/1108:Tm50CC2F3/MRH106:Tm50CIncorporatingmolecularidentificationofMeloidogynespp.intoalarge-scaleregionalnematodesurveyTOPowers,PGMullin,TSHarris,LASutton...-Journalof…，2005-...ForM.chitwoodiidentification,thefirstoftwoPCRamplificationswasconductedwithprimersetC2F3/1108(5GGTCAATGTTCAGAAATTTGTGG3and5TACCTTTGACCAATCACGCT3)locatedintheCOIIand16Sribosomalmitochondrialgenes,respectively(Powersand..Biometrical,biochemical,andmoleculardiagnosisofPortugueseMeloidogynehispanicaisolatesCMMaleita,MJSimdes,CEgas,RHCCurtis...-Plant2012-AmPhytopathSociety...MitochondrialDNAfromisolatesPtHi3ofM.his-panicaandItEofM.ethiopicaweresequeneedwiththeprimersetC2F3(5'-GGTCAATGTTCAGAAATTTGTGG-3')andMRH106(5'-AATTTCTAAAGACTTTTCTTAGT-3')locatedintheCOIIgeneandthe16SrRNA...[pDF]Prevalenee,incideneeandmolecularidentificationofroot-knotnematodesoftomatoinPakistanMAhmed-AfricanJournalofBiotechnology,2012-academicjour...DNAamplificationofmtDNAwithC2F3/1108primersyieldeda1700bpsizeproductforallthreespeciesofRKNsincomparisonwith520and750bpforM.chitwoodiandenterolobii,respectively,whichwereutilizedascontrol.Plant-parasiticnematodesinsugarcanefieldsinKitadaitoIsland(Okinawa),Japan,asapotentialsugarcanegrowthinhibitorMKawanobe,NMiyamaru,KYoshida...-...,2014-...NematodeidentificationusingDNAsequeneedataSinglenematodeswerehandpickedusingasterilisedneedle,rinsedwithdistilledwaterandplacedonaglassslide.???F194(5-CGTAACAAGGTAGCTGTAG-3)andF195(5-TCCTCCGCTAAATGATATG-3)wereuLdtihiH wunuui(ein'piaicDhAwaz；'IKFU5SttFig.1-I/xalioJlEtratMM血柯f珅』I'DNAyflheirtemdtian沉ribed O皿勇uittlin[lij&study”Thery：gioji5ihhuiwccnIhsrDNA{IBS.S.8Suud閤時arctheITS.TheIBSandZ85genesueimneated.□Detectionofthepinewoodnematode,Bursaphelenchusxylophilus,usingareal-timepolymerasechainreactionassayAXCao,XZLiu,SFZhu,BSLu-Phytopathology,2005-AmPhytopathSociety...Onetofournematodeswereplacedinto15ylofdouble-distilledwateron...sterile0.5-mlthinwalledPCRtubecontaining8ylofnematodelysisbuffer...Genus-specificprimersF194(5'-CGTAACAAGGTAGCTGTAG-3')and5368(5'-TTTCACTCGCCGTTACTAAGG-3')wereTable1Primers^"昶討inthepresetstmfy.PrimercodeSequence(JJf3r)AmplifiedoencReferencesTW81GTTTCCGT.XGGTCAACCTGCITSrRNA'larihaMaatieid/.AB28AIAT<X：mAGTTCAGCGGCTFlQdCGTAACAAGGT^GCTGT^GGITS-rRNAPly.51vciTCCGClAAAlGALAJ'ACAA￡；TACCCTC；AGGliAAAtlTKJD2-D8of2SSrftNASabbolinetaLD5BTCGGAAGGAACCAGCTACLXU8JIAAYY^RACMAAGOCNTYTGGAChsp90Stance&Gand(2005)I1110TCRCARTTVTCC.^TGATRAAVACTW&1GTTTCCGTAGGTGAACCTGCITS-j-EiNATliiisiudjsruup-spccLiicCTIAAGCC.\HjTGCCAACTCTWB]GnTCCGT.XGGTGAACCTGCITS-rEiNAThisstudyspcxTics-spccilicGTGCACT3.ATCTTATEYTGTXTGGDeLucaF,TroccoliA,DuncanLW,etal.Pratylenchusspeijerin.sp.(Nematoda:Pratylenchidae),anewroot-lesionnematodepestofplantaininWestAfrica.Nematology,2012,14(8):987-1004.IGSsequeneevariation,group-1intronsandthecompletenuclearribosomalDNAoftheentomopathogenicfungusMetarhizium:excellenttoolsforisolatedetection…MPPantou,AMavridou,MATypas-FungalGeneticsandBiology,2003-Elsevier...18SF,GCGAAACTGCGAATGGCT,Thiswork.18SR,GTAATGATCCCTCCGCTG,Thiswork.TW81,GTTTCCGTAGGTGAACCTGC,Curranetal.(1994).AB28,ATATGCTTAAGTTCAGCGGGT,Curranetal.(1994).Ma-ITS2,GTCCACTGCCGTAAAACCCC,Thiswork.Heteroderavallicolasp.n.(Tylenchida:Heteroderidae)fromelmtrees,Ulmusjaponica(Rehd.)Sarg.inthePrimorskyterritory,theRussianFarEast,withrDNA…ASEroshenko,SASubbotin...-RussianJournalof 2001-...DNAfragmentsweresequeneedinbothdirectionswithTW81,AB28,5.8SM2(5'-CTTATCGGTGGATCACTCGG-3')or5.8SM5(5'-GGCGCAATGTGCATTCGA-3')primerswithaBigDyeTerminatorCycleSequencingReadyReactionKit(PEAppliedBiosystems,UK)Nematodeuniversalprimers:GymnodiniumnolleriEllegaardetMoestrupsp.ined.(Dinophyceae)fromDanishwaters,anewspeciesproducingGymnodiniumcatenatum-likecysts:molecularand…MEllegaard,YOshima-Phycologia,1998-...1989)conservedpositions708-727(D3A;5'GACCCGTCTTGAAACACGGA-3')and1011-992(D3B;5'TCGGAAGGAACCAGCTACTA-3').Double-strandedandsingle-strandedPCRamplificationswereperformedinathermocyclerwithaninitialdenaturationstepof3minatNuc