采用实时荧光定量PCR技术比较提取土壤真菌DNA方法的差异王淑芳

PCRDNA1212*111．2220052．222005PCROMEGADNADNA。5Phytophthoracapsici。OMEGADNAPCR。DNAPCRDNA。2．78。OMEGADNADNA。。PCRDNAS151．9A0517－6611201133－20318－04ComparisonoftheDifferenceintheFungusDNAExtractedwiththeArtificialMethodandKitMethodWANGShu-fangetalHuaihaiInstituteofMarineCollegeLianyungangJiangsu222005AbstractObjectiveThedifferenceintheisolationoffungusDNAwithartificialandKitextractingmethodwascomparedbasedonthetestofthereal-timequantitativefluorescesPCRtechnique．MethodThehyphaeofPhytophthoracapsiciwithfivedifferentconcentrationsandtheCKwasinoculatedinthesterilesoilmediumandtheDNAfromsoilsampleswasextractedwithartificialandOMEGADNAkitrespectivelyandthenthePhytophthoracapsiciwasquantitativelydetectedwithreal-timequantitativePCRmethodanditsspecificprimers．ThecharacterofthetwoisolationDNAmethodswasanalyzedbasedonthecomparisonoftheresult．ResultItwasfoundthatthesoilfungusDNAwaslittleaf-fectedbyothermaterialsinthetwomethodsandthetemplateDNAofreal-timePCRtechniquecouldbeformed．ThequantitativeresultsofPhytophthoracapsiciobtainedbyKitmethodwas2．78timeshigherthanthatobtainedinaverage．ConclusionThekitmethodextractedmoreDNAanditsquantitativeresultsweremoreaccurateallreagentsneededbyartificialmethodwerecommonbiochemicalreagentswhichwereaffordableandeasytoacquire．Thereforebothmethodshadtheirownadvantagesthechoiceofthemshouldbebasedonpracticalconditions．KeywordsReal-timequantitativefluorescesPCRFungusDNAinsoilCARS-25-B-2Z2009044。[e-mail]。*[e-mail]。2011-08-15DNA。DNA。DNADNA1－7。DNADNA。TNEP-PBS6。、、、1－37、、K13－57。DNA。PCRDGGE。PCRDNA。DNA4PCRPhytophthoracapsiciDNA。11．11．1．1。。。1．1．2。PCRSYBRPremixExTaqTMPerfectRealTimeSigmaAR。DNABioTek。PCR。DNAOMEGA。1．1．3。Bio-RadiQ5PCRAmershamUltro-spec3300pro。1．21．2．1。1．2．1．1。80℃200。2h。1．2．1．2。PD25℃、150r/min2d4。10000r/min5min2．4g7．6mlJournalofAnhuiAgri．Sci．2011393320318－2032112ml16ml22ml26ml32ml36ml42ml46ml5。1．2．1．3。6。1～560．5g50.00、12.50、3．13、0.780.20mg/g。。1．2．2DNA。1．2．2．1LabMethod。3DNA4。CTAB-SDS0．5g10ml3mlTENPBufferTris50mmol/LEDTA20mmol/LNaCl100mmol/LPVP10g/LpH10．010min12000r/min5min3mlTENPBuffer23mlPBSBufferNaCl8g/LKCl0．2g/LNa2HPO41．44g/LKH2PO40．24g/LpH7．410min12000r/min5min3g0．5cm2ml2%CTABNaCl1．4mol/LTris-HCl100mmol/LpH8．0EDTA20mmol/LpH8．02%CTAB10min37℃1h15min35min1．5ml3%SDS5min65℃2h20min38000r/min15min/25∶241015000r/min15min0．1NaAc3mol/L0．61h15000r/min20min70%2150μlDNA。DNADNA3DNAPCR。1～6。1．2．2．2OMEGADNA。3DNA。0．5g、500g1mlBufferSLXMlus3～5min100μlBufferDS70℃10min3000r/min3min2ml270μlBufferSP25min13000r/min、4℃5min2ml0．720～30－20℃1h13000r/min、4℃10min200μlElutionBuffer10s65℃10～20min50～100μlHTRReagent10s2min13000r/min2min2mlXP2Buffer10000r/min1min2ml300μlXP2Buffer10000r/min1min2ml700μlSPWWashBuffer10000r/min1min12ml13000r/min2min1．5ml30～100μlElutionBuffer65℃10～15min13000r/min1minDNA。3DNA1’～6’。DNA260nmA260nmA260nm/A280nm。1．2．3PCR。PCRSYBRGREENI。P1-F5’-AGGTGCACACAAAC-TTCCC-3’P1-R5’-GCAAAGCCGATTCAAAAG-3’。He8。PCRSYBRbuffer10μl0．3μmolDNA1μl1～10ng20μl。3DNA。95℃30s95℃5s60℃30s40iQ595℃60s55℃60s55℃30s。1．2．4。PCRiQ5Bio-RadiQ5OpticalSystemSoftware。22．1DNAOMEGADNA3DNA5μl1%。1DNA3DNA0．780.20mg/g2DNA。350.00、12.50、3.13mg/g。DNA。OMEGADNA5DNA130．780．20mg/g。66’DNA。2．2PCRITSDNA101．000×109～1．000×102/μl。“1．2．3”PCRPCR1μl1～61’～6’DNA。3Y=－3．295X+44．485R20．9801．000×109～1．000×1028。－3．295913023933PCRDNAM1Takaraλ-HindⅢdigestDNAMarker23130bpM2Tranks2KDNAMarker2000bp1～61～6DNA。NoteM1．Takara-λHindⅢdigestDNAMarkerthelargestbandwas23130bpM2isTranks2KDNAMarkerthelargestbandwas2000bp1－6．Soilsamplesfrom1to6．1DNAFig．1SoilDNAisolatedbylabmethodMTakaraλ-HindⅢdigestDNAMarker23130bp1～61’～6’DNA。NoteM．Takaraλ-HindⅢdigestDNAMarkerthelargestbandwas23130bp1～6．Soilsamplesfrom1’to6’．2OMEGADNAFig．2SoilDNAisolatedbyOMEGAkitmethod101．1%2．01PCR2PCR。3PCRFig．3Standardcurveofreal-timePCRofP．capsiciCt。1DNA3．87±0．19×104/μl2．78±0．13×102/μl。OMEGADNA9．51±0．47×104/μl1．01±0．06×103/μl。66’PCR。DNA。OMEGADNADNA52．46、2．56、3．09、2．173．632．78。DNA70%～80%OMEGADNA1．35～1．53。1PCRTable1Resultsofreal-timePCRbylabmethodandkitmethodMyceliumamount∥mglglgmyceliumamountLabextractionNo．Quantitativeresultscopy/μllglgMeanofquantitativeresultsOMEGAKitextractionNo．Quantitativeresultscopy/μllglgMeanofquantitativeresults50．001．69913．87±0．19×1044．5881'9．51±0．47×1044．97812．501．09721．67±0．09×1044．2232'4．27±0．01×1044．0293．130．49634．01±0．22×1033．6033'1．24±0．04×1043．4910．78－0．10947．36±0．40×1023．4614'1．60±0．09×1033．2040．20－0．69952．78±0．13×1023．4445'1．01±0．06×1033．00406－－6'－－1～41’～4’DNA。4、51～41’～4’DNA。DNAY=0．6639X+3．4405R20．9526OME-GADNADNAY=0．9724X+3．1517R20．9389。DNA9PCRDNA。4、5OMEGADNAOME-GADNADNA023022011。DNA。4DNAFig．4StandardcurveofsoilDNAextractionbylabmethod5OMEGADNADNAFig．5StandardcurveofsoilDNAisolationbykitmethod3DNADNA。DNA。DNADNA。DNA。OMEGADNADNAPCROMEGADNAPCRDNADNADNADNA2．78。。。1．DNAJ．20092299－302．2．DNAJ．200811703－705．3．DNAJ．20074611－616．4．DNAJ．20055854－860．5．DNAJ．200839112－16．6．TENP－PBSDNAJ．2010382412978－12980．7．DNAJ．20098151－155．8HEJZSHENJPZHANGLMetal．Quantitativeanalysesoftheabun-danceandcompositionofammonia-oxidizingbacteriaandammonia-oxidi-zingarchaeaofaC