乳酸杆菌及嗜热链球菌的种水平鉴定-16SrRNA基因PCR扩增及序列分析

45439420107JOURNALOFHYGIENERESEARCHVol.39No.4Jul.20101000-8020201004-0454-06··———16SrRNAPCR12310002116SrRNAPCR。DNAPCR16SrRNAPCR。16SrRNAPCR50724、12、7、3、2、11。16SrRNAPCR、。16SrRNAPCRR155.51TS207.4AIdentificationofLactobacillusandStreptococcusthermophilusbyPCRamplificationandsequenceanalysisof16SrRNADONGYinpingCUIShenghuiLIFengqinYUHongxiaInstituteforNutritionandFoodSafetyChinaCDCBeijing100021ChinaAbstractObjectiveTodevelopaPCRmethodforidentifyingthe16SrRNAofLactobacillusandStreptococcusthermophilusatthespecieslevel.MethodsOptimizingthemethodforDNAextractionandtheconditionsforPCRamplification.JoiningthePCRamplificationproductsfrom16SrRNAtoplasmidpuc18-Tanddetectingthesequence.ResultsAll50isolatesrecoveredfromyoghourtproductswerecharacterizedby16SrRNAsequenceanalysisand7groupswereidentifiedasL.bulgaricus24strainsS.thermophilus12strainsL.acidophilus7strainsL.casei3strainsL.delbrueckii2strainsL.fermentum1strainandS.lutetiensis1strain.Conclusion16SrRNAPCRmethoddevelopedinthisresearchisasensitiveandreliablemethodfortheidentificationofbothLactobacillusandStreptococcusthermophilus.KeywordsLactobacillusStreptococcu[e-mail]3。。。。、、、、、。、、polymerasechainreactionPCRrRNA。16SrRNAPCRDNA16SrRNA16SrRNAGENBANK。16SrRNA/DNA1-2。16SrRNAPCR。4.16SrRNAPCR45511.11.1.1Lactobacilliuscaseisubspcasei1.2435、Lactobacilliusdelbrueckiisubspbulgaricus1.2161、Lactobacilliusplantanum1.2158、Lactobacilliusacidophilus1.2686。1.1.250。1.1.3MRSMHBectonDickinsonSOB20g、5g1000ml5mol/LNaCl2ml、3mol/LKCl0.83ml1000ml121℃15min。SOCSOB100ml1mol/L2ml。Terrific12g、24g、9.4g、2.2g1000ml1000ml121℃15min。1.1.4EBSigmaDNAMarker、、OXOID、、、、、ServaIPTG、X-Gal、pUC18-T、DNTP、DNATaKaRaQuant-ItTmdsDNAHSAssayKitsInvitrogen、TaqDNA、10×TaqFERMENTASM13TaKaRaPCRBIOBASIC。1.1.54±1～50±1℃SANYOBiomerieuxBECKMAN、PCRBIO-RAD10～100℃-。1.21.2.1DNAMRS36±1℃48hMRS36±1℃24h1.5ml2ml3000×g10minDNADNADNAQuant-ItTmdsDNAHSAssayKits－20℃。1.2.21.2.2.1PCR50μl10×Taq5μlMgCl24μldNTP1μlFD15′-AGAGTTTGATCCTGGCTCAG-3′RP25′-ACGGCTACCTTGTTACGACTT-3′10.4μlTaqDNA0.3μl1.5UDNA2μl50μl。1.2.2.2PCRDNAPCR4℃。95℃4min95℃1min50℃～55℃55、54.8、54.2、53.3、52、51.1、50.5、50℃1min72℃1min3072℃4min。95℃4min95℃1min55℃1min72℃1min3072℃4min。95℃4min95℃1min50℃1min72℃1min3072℃4min。95℃4min95℃1min50℃1min72℃1min1055℃1min72℃1min3072℃4min。1.2.2.3PCR5μlPCR5μl100～2000bpmarker2%EB0.5μl/ml。PCREZ-10SpinColumnPCR。1.2.3PCR2。1.2.4PCR1.2DNAM131.2.2.1PCR95℃4min95℃1min55℃1min72℃1.5min3072℃4min。1.2.2.3PCR。22.1DNA1DNADNAPCRDNA。DNA。2.2PCR2PCR53.6℃、53℃55℃。PCR50℃50℃1055℃30PCR。2.350GENBANKhttp//rdp.cme.msu.edu/1。116SrRNAPCR50724、12、7、3、2、11。16SrRNA。MEGA3.13。4563915016SrRNATable116SrRNAgenesequencinginidentificationof50strainsofbacteriaisolatedfromyogurt16SrRNA1%1－1L.bulgaricus1001－2L.bulgaricus1002－1L.bulgaricus1002－2L.acidophilus1003－1L.fermentum1003－2S.lutetiensis1004－1L.bulgaricus1004－2L.acidophilus1005－1L.delbrueckii1005－2L.delbrueckii1006－1L.bulgaricus1006－2L.bulgaricus1007－1L.bulgaricus1007－2L.bulgaricus1008－1L.bulgaricus1008－2L.bulgaricus1009－1L.bulgaricus1009－2L.bulgaricus10010－1L.bulgaricus10010－2S.thermophilus10011S.thermophilus10012L.bulgaricus10013－1L.bulgaricus10013－2S.thermophilus10014－1L.bulgaricus10015S.thermophilus10016S.thermophilus10017－1L.bulgaricus10017－2L.bulgaricus10018－1L.casei10018－2L.bulgaricus10019L.acidophilus10020L.acidophilus10021L.acidophilus10022L.acidophilus10023－1L.bulgaricus9823－2L.bulgaricus10024－1L.casei10024－2L.casei10025－1L.bulgaricus10026－1S.thermophilus10026－2S.thermophilus10027－1L.bulgaricus10027－2S.thermophilus10028－1S.thermophilus10028－2S.thermophilus10029－1S.thermophilus10029－2S.thermophilus10030L.acidophilus10031L.bulgaricus9714.16SrRNAPCR45712～5DNAPCR6～9DNAPCR263748591DNAPCRFigure1ComparisonoftwoDNAextractionmethodsforPCRamplificationproductsA850℃、50.5℃、51.1℃、52℃、53.3℃、54.2℃、54.8℃、55℃2～911～181、10B55℃1～57～196C50℃D50℃10、55℃302PCRA～DFigure2ElectrophoresisresultsofPCRsegmentofLactobacillusandStreptococcus.thermophilusindifferentannealingtemperaturesA-D16SrRNA23Ⅰ、Ⅵ、Ⅸ、Ⅹ413Ⅲ、Ⅴ、Ⅶ、Ⅷ412Ⅵ。Ⅲ。Ⅰ871。31。316SrRNADNA16SrRNA16SrRNA、。16SrRNAPCRPCR、Mg2+、、。PCR。FD1、RP24583935016SrRNAFigure3Cladogramof16SrRNAgenesequencesof50stainsofbacteriaisolatedfromyogurt16SrRNAPCRDNADNAPCR。55℃50℃1055℃30。。DNAPCR。3－216SrRNA16SrRNA1。16SrRNA4654.465、。。。。。。。1.J.200128916-17.2.2002J.2006236457-465.3.J.2002364278-279.4FAWELLJBAILEYKCHILTONJetal.Fluorideindrinking-waterR.WHO200637.5HealthCanada.FluorideDB/OL.2009-02-06.http//。。16SrRNA16SrRNA。。。。。3。、4。1WEISBURGWGBARNSSMPELLETIERDAetal.16SribosomalDNAamplificationforphylogeneticstudyJ.Bacteriol1991173697-703.2.16SrDNAJ.200717111963