不同破壁方法提取真菌DNA的比较-周万青

:1001-764X(2009)03-0212-03:R446.6:ADNA*周万青1,2,李芳秋2,王立魁2,邵海枫2,史利宁2(1.,212013;2.,210002):探索和评价提取致病性真菌DNA的简便方法分别用6种方法(液氮冻融-70冻融溶胞酶消解超声破碎高盐溶液尿素溶液)裂解真菌细胞壁,再用市售DNeasyBlood&TissueKit试剂盒提取DNA,最后通过对提取物进行比色定量和PCR检测评价各种提取方法的优弊酶消解破壁法提取真菌DNA效率最高,重复性良好模拟全血标本中白色念珠菌检测限为102个孢子,烟曲霉及新型隐球菌检测限为103个孢子酶消解破壁法提取真菌DNA在6种方法中效率最高,可用于临床常见真菌感染的PCR快速诊断:真菌;DNA;提取;PCR,,:[1]-70[2][3][4][5][6]PCR,6DNA,11.1TaqDNAdNTPs(Promega),K(Merck),(lyticase)(Sigma),DNeasyBlood&TissueKit(Qiagen),(Amersco),(Serva),BioDL-2000DNAMarker();(JY-250),PCR(Eppen-dorf),()1.2:(C1)()(A1,)1.3,3072h,;,3072h,(9g/LNaCl),,(1~5)109/ml103/ml~107/ml10l500l,101~10530mg,DNA1.4500l1.5mlEP,1ml(10mmol/LTris-HClpH7.6,5mmol/LMgCl2,10mmol/LNaCl),3710min,5000g10min,,500l(10mmol/LTris-HClpH7.6,10mmol/LEDTA,50mmol/LNaC,l2g/LSDS,200g/mlK),652h,5000g15min,6DNA1.5DNA1.5.1600l(1mol/L,100mmol/LEDTA,14mmol/L-),200U,3030min,300g10min,180lATL(Qiagen)1.5.2600l,180w60s1.5.3-70600l,-70(95%)10min,,31.5.4600l,2122009273ChineseJournalofClinicalLaboratoryScience,2009,Vol27,No3*:(06Z43):,1981,,,:,E-mai:[e-mail]2min,951min;2,30s,10min1.5.5200l(4mol/L,50mmol/LTris-HClpH8.0,5mmol/LEDTA,0.1mol/L-,5g/L),5min,12000r/min1min,,1ml2,600l1.5.6600l(8mol/L,0.5mol/LNaC,l20mmol/LTris-HClpH8.0,20mmol/LEDTA,20g/LSDS),3h,12000r/min1min,,1ml2,600l:20lK(Qiagen),552h,DNeasyBlood&TissueKit(Qiagen),100l31.6DNA30mg,6,DNA1.7PCR18SrDNA[7]::5-GATACCGTCGTAGTCTTA-3,:5-ATTCCTCGTTGAAGAGC-3,580bp,20:lDNA5,l10buffer(Mg2+)2,l1UTaq,10mmoldNTP,37.5mmolMgCl2,2.5pmol:945min,9430s,5230s,7230s,35,727minDNA,5l20g/L,,1.8SPSS10.0,t22.16DNA6,30mgDNA,DNA1DNA,A260/A28030mgDNA14.2g,(P0.05),5g,DNA1DNA2.2PCRDNA6,102,7.7h;103,7.2h;104,10h;-70105,8h;105,7.4h;,7.3h2:M,DNAmarker;1,;2~6,105~101;7,;8,;A~F-7062PCRDNA2.3PCRDNA,,10336QiagenDNA,,,-70,,,DNA,,2132009273ChineseJournalofClinicalLaboratoryScience,2009,Vol27,No3,DNA6DNA,,,QiagenDNA,PCR,,102;,103,;,,104,;-70105;,Griffiths[8]DNA,PCR103:QiagenDNA,,,EP,,3,PCR,:[1]ManianS,SreenivasaprasadS,MillsPR.DNAextractionmethodforPCRinmycorrhizalfungi[J].LettApplMicrobio,l2001,33(4):307-310.[2]GriffinDW,KelloggCA,PeakKK,etal.ArapidandefficientassayforextractionDNAfromfungi[J].LettApplMicrobio,l2002,34(3):210-214.[3]WilliamsonEC,LeemingJP,PalmerHM,etal.Diagnosisofinva-siveaspergillosisinbonemarrowtransplantrecipientbypolymerasechainreaction[J].BrJHaemato,l2000,108(1):132-139.[4]HauglandRA,HeckmanJL,WymerLJ.EvaluationofdifferentmethodsfortheextractionofDNAfromfungalconidiabyquantitativecompetitivePCRanalysis[J].JMicrobiolMethods,1999,37(2):165-176.[5]DanilevichVN,GrishinEV.Anewapproachtotheisolationofge-nomicDNAfromyeastandfung:ipreparationofDNA-containingcellenvelopesandtheiruseinPCR[J].BioorKhim,2002,28(2):156-167.[6]SansinforianoME,PadillaJA,HermosodeMendozaJ,etal.RapidandeasymethodtoextractandpreserveDNAfromCryptococcusneo-formansandotherpathogenicyeasts[J].Mycoses,1998,41(5-6):195-198.[7]VanBurikJA,MyersonD,SchreckhiseRW,etal.PanfungalPCRassayfordetectionoffungalinfectioninhumanbloodspecimens[J].JClinMicrobio,l1998,36(5):1169-1175.[8]GriffithsLJ,AnyimM,DoffmamSR,etal.ComparisonofDNAex-tractionmethodsforAspergillusfumigatususingrea-ltimePCR[J].JMedMicrobio,l2006,55(Pt9):1187-1191.ComparisonoflysismethodsforfungalDNAextractionZHOUWan-qing1,2,LIFang-qiu2,WANGL-ikui2,etal.(1.SchoolofMedicalTechnology,JiangsuUniversity,Jiangsu,Zhenjiang212013;2.InstituteofMedicalLaboratorySciencesofPLA,NanjingGeneralHospitalofNanjingMilitaryCommand,PLA,Jiangsu,Nanjing210002,China)Abstract:ObjectiveToinvestigateandevaluatesimpleDNAextractionmethodforfungaldiagnosis.MethodsSixcelllysismethodsin-cludingfreezethawinginliquidnitrogenorat-70,enzymedigestion,sonication,chaotropicsalts,andureabufferwereinvestigated.EachlysisprocedurewasfollowedbyDNApurificationusingtheQiagenkit.TheefficiencywasestimatedbyquantitationandPCR.Re-sultsOfallsixextractionmethods,enzymedigestioncombinedwiththekitresultedinthehighestyield.ThedetectionlimitforCand-idaalbicans,Aspergillusfumigatus,andCryptococcusneoformansinsimulatedbloodsampleswithuniversalprimerPCRwas102,103,and103cellsperreactionrespectively.ConclusionsEnzymedigestioncombinedwiththekitforextractingDNAfromfungiisusefulfordiagnosingfungalinfection.Keywords:funga;lDNAextraction;PCR(:2008-10-28)(:杨林)2142009273ChineseJournalofClinicalLaboratoryScience,2009,Vol27,No3