实时荧光定量PCR技术的原理及其应用研究进展

16420078CHINESEJOURNALOFHISTOCHEMISTRYANDCYTOCHEMISTRYVol1161No.4Aug.2007PCR3(,,,100069)PCR;;DNAQ52311A20062102312007201226,(1975),,3(Towhomcorrespondenceshouldbeaddressed)1983Mullis(polymerasechainreaction,PCR),PCRPCR,,(fluorescenceresonanceenergytransfer,FRET)PCR,[1]PCR(real2timefluoro2geneticquantitativePCR,FQ2PCR)PCRPCR,PCR,DNA,,[2]11PCR1111992HiguchiPCR,FRET,,,()(),,FRET5(R),3(quencher,Q),(7210nm),,S/N(2,signal2to2noiseratio)112PCR(threshold):PCR15,3-1510Ct:,CCycle,tthresholdCtPCRPCR,,,CtRn:DNA(1),CtDNA(Rn)PCR4(2)1PCRFig.1TheimportantparametersofrealtimePCR2PCR4Fig.2The4phasesofrealtimePCR©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[3]PCR,SYBRGreenIDNA;211,,TaqManTM()TaqmanMGB()(Dual2oligoFRETpairs()Molecularbeacons()ScorpionsTM/AmplifluorTM()Univer2salprobelibraryLNA(lockednucleicacids()Lux(lightuponextention)Simpleproble[4]21111TaqMan20-24bp,5,3,DNATaq53,,PCR,Taq53,,,,,PCRPCR,FRET,PCR,Taq53,,,DNA,,PCR,RnRnCt,Ct,FAMTETVICHEX(3)3TaqManFig.3TaqManProbe21112TaqmanMGBTaqmanMGBTaqman,3MGB(minorgroovebinder),DNA;MGBTM,1:,;,;MGB,SNP(4)4TaqManMGBFig.4TaqManMGBprobe21113RochePCR,FRET53,,(1-5),FRET,,PCR,,;,,,LC2Red640LC2Red705(5)5Fig15Two2hybridprobe3944.PCR©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(molecularbeacon),,15-30;,,5-7;35,,FRET,,,PCR,DNA,,,,,,(6)FAMTexasRed6Fig16Molecularbeacon21115AmplisensorBiotrnicsTechPCR,;,,,PCR,,,Amplisensor,,PCR21116Universalprobelibrary2LNA(lockednu2cleicAcids)Roche,89,,89;7000LNARNA,2p24p2,5p3p;LNATm,PNA(PNAS2000,97:563325638)1LNADNADNADNADNARNAPNADNA,;,,,LNA;PCR(7)7LNAFig17LNAstructure21117LUXLUX(lightuponextention,LUX);,Ct,LUX,,,(8)8LuxFig18ThefunctionmechanismofLux21118SimpleRoche,5,5linker,,,linker,,(9)49416©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(dsDNA)PCRdsDNA,SYBRGreenILCGreenTMI[5]21211SYBRGreenI,DNA,,dsDNA,,,DNA,SYBRGreen,,,dsDNA,PCRPCR,,,(10)10SYBRgreenFig110SYBRGreenI21212LCGreenTMIdsDNA,SYBRGreenI,,dsDNA,PCR,LCGreenTMIPCRPCRLCGreenTMI,,,,PCR(Tm),(SNP)LCGreenTMI,31PCRPCR,:;CT,DNA311FQ2PCR,;PCR;PCR,,,[6]DNARNA,PCR,Ct,,Ct,,RNA,RNA,cDNAPCR,,,,260nm,,312,PCR,GAPDH22rRNA[7]2actin,,,,,,,,1,n,5944.PCR©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[8]313CtCT,,2,,CT,CTPCR,PCRCt,,PCRCt,(Ct=1)2DNACTDNACT,,,[9];,41PCRPCR,,SNP;[10]411,,[11]41111,,,dsDNASYBRGreenI,2[12],,PCR()mRNA,,,,[13],PCR41112(SNP)PCR[14],,,,,,,,(Tm)41113TessonPCR,,,[15],PCR,412,,PCRhTERTERMDR1,,[16]69416©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(CEA)mRNA,[17],,,,PCR[18]EckertPCR,413,EB[19],2004SarsPCRPCR,,,,PCR(HIV),51,,FQ2PCR,,;,[20]FQ2PCR,1CleggRM1Fluorescenceresonanceenergytransfer1CurrOpinBiotechnol,1995,6(1):10321102MikaelKA,JoseMA,MartinB,etal1Thereal2timepoly2merasechainreaction1MolecularAspectsofMedicine,2006,27:95$1253JonesCD,YeungC,ZehnderJL,etal1Comprehensivevalidationofareal2timequantitativePCRassayforclinicallaboratoryuse1JClinPathol,2003,120(1):422484WolfgangK,JonathanN,KarasS,etal1Fluorogenicprimersforreal2timePCR1AmericanBiotechnologyLabo2ratory,2003,(7):522565WittwerCT,ReedGH,GundryCN,etal1High2resolutiongenotypingbyampliconmeltinganalysisusingLCGreen1ClinChem,2003,49(6pt1):85328606ZimmermannB,HolzgreveW,WenzelF,etal1Novelre2al2timequantitativePCRtestfortrisomy1ClinChem,2002,48(2):36223637GalbiatiS,SmidM,GambiniD,etal1FetalDNAdetec2tioninmaternalplasmathroughoutgestation1HumGenet,2005,117(223):24322488RobertMW,StellaL,MichaelG,etal1MeasurementofEpsteinbarrvirusDNAloadsinwholebloodandplasmabyTaqManPCRandinperipheralbloodlymphocytesbycompetitivePCR1JClinMicrobiol,2003,41:5245252499MagnusL,CharlesH1DynamicrangeandreproducibilityofhepatitisBvirus(HBV)DNAdetectionandquantifica2tionbycobasTaqmanHBV,areal2timesemiautomatedas2say1JClinMicrobiol,2005,43:42512425410NathalieD,AxelleD,VroniqueS,etal1Quantifica2tionofHumanimmunodeficiencyvirustype1proviralloadbyaTaqManreal2timePCRassay1JClinMicrobiol,2001,39:13032131011HwaHL,KoTM,YenML,etal1Fetalgenderdetermi2nationusingreal2timequantitativepolymerasechainreac2tionanalysisofmaternalplasma1JFormosMedAs2soc,2004,103(5):364236812,,,11,2005,28(8):858286113UenoH,YoshidaK,HiraiT1QuantitativedetectionofcarcinoembryonicantigenmessengerRNAintheperito2nealcavityofgastriccancerpatientsbyreal2timequanti2tativereversetranscriptionpolymerasechainreaction1AnticancerRes,2003,23(2C):17012170814NieuwenhuisEJ,JasparsLH,CastelijnsJA,etal1Quantitativemoleculardetectionofminimalresidualheadandneckcancerinlymphnodeaspirates1ClinCancerRes,2003,9(2):755276115LiewM,PryorR,PalaisR,etal1Genotypingofsinglenucleotidepolymorphismsbyhigh2resolutionmeltingofsmallamplicons1ClinChem,2004,50(7):11562116416PietA,vanRijn,GerardJW,etal1Detectionofeco2nomicallyimportantvirusesinboarsemenbyquantitativeRealTimePCRtechnology1JournalofVirologicalMeth2ods,2004,120:151216017AndersenCL,JensenJL,Orn