CYP2C19基因多态性检测方法的建立-黄杰

*[e-mail][e-mail]2C19112211*1．1000502．430075CYP2C19PCＲ－CYP2C19。PrimerExpress3.0CYP2C19CYP2C19*2、CYP2C19*3CYP2C19*17。、DNAgenomicDNAgDNAPCＲCYP2C19PCＲ。gDNA、。100Kappa。CYP2C19PCＲ－CYP2C19*2、*3*171nggDNA。Kappa1.00。CYP2C19、CYP2C19。P450S－－4－CYP2C19PCＲ－Ｒ917A0254－1793201509－1562－06doi10.16155/j．0254－1793.2015.09.09EstablishmentofCYP2C19polymorphismdetectionmethodHUANGJie1YUTing1ZHANGZhe2CAICong－li2GAOShang－xian1QUShou－fang1*1．NationalInstitutesforFoodandDrugControlBeijing100050China2．WuhanYZYMedicalScience＆TechnologyCo．LtdWuhan430075ChinaAbstractObjectiveToestablishfluorescence－PCＲprobemethodforCYP2C19genepolymorphismdetectionandprovideanewdetectionkittopredicttheefficacyofdrugmetabolismthroughCYP2C19．MethodsCYP2C19genesequenceCYP2C19*2CYP2C19*3andCYP2C19*17wasselectedastarget．TheprimersandprobesweredesignedwithPrimerExpress3.0software．Firstheterozygousmutantandhomozygousmutantandwild－typesampleswereconfirmedbysequencingusingthehumanwholebloodgenomicDNAgDNAastemplates．Therightcombinationofprimersandprobeswereselected．ThenthefluorescencePCＲmethodforCYP2C19genepol-ymorphismdetectionwasestablishedandoptimizedwiththeaccuracysensitivityandspecificityoffluorescencePCＲmethodwithgDNAthespecificconcentrationandgenotypeknownasqualitycontrol．Finallytheclinicalapplicabilitywasverifiedwith100casesofclinicalsamples．TheresultswerecomparedwiththedirectsequencingandtheKappavaluewascalculated．ＲesultsFluorescencePCＲmethodforCYP2C19genepolymorphismdetec-tioncanaccuratelydetectthedifferentpolymorphicvariantsofCYP2C19geneincludingheterozygousmutantandhomozygousmutant．ThesensitivityoffluorescencePCＲdetectioncanreachto1ng·reaction－1．TheresultsofclinicalsamplesshowgoodconsistencywiththeresultsofsequencingandtheKappavalueis1.00．ConclusionFluorescencePCＲmethodforCYP2C19genepolymorphismdetectionprovedtobeaneffectivemethodwithbetter—2651—ChinJPharmAnal2015359accuracysensitivityspecificityandclinicalapplicabilityandcanbeappliedtopredicttheefficacyofdrugsme-tabolismthroughCYP2C19．KeywordsgenepolymorphismtestingcytochromeCYPS－mephenyltoin4－hydroxylaseCYP2C19fluores-cencepolymerasechainreactionPCＲ－fluorescentprobemethoddrugefficacytestingP450cytochromeP450CYP450。CYP2C19S－－4－CYP450、、1。CYP2C1910q24.1－24.3CYP2C19*1/*1。CYP2C19*25G681ACYP2C19*34G636A2。CYP2C19*2CYP2C19*3CYP2C193。99%CYP2C19*2CYP2C19*3。2006Sim4CYP2C19CYP2C19*17806C＞T。CYP2C19*17CYP2C19*17CYP2C195。CYP2C19、6－7PCＲ8－9。CYP2C19PCＲ－CYP2C19CYP2C19*2、CYP2C19*3CYP2C19*173CYP2C19。15xPCＲbufferdNTPmixTaqDNATakara。DNA。In-vitrogen。AppliedBiosystems。CYP2C19*2、CYP2C19*3CYP2C19*172CYP2C192100。22.1PrimerPremier5.0PrimerExpress3.0CYP2C19*2、CYP2C19*3CYP2C19*17SNP。glyceraldehyde－3－phosphatedehydro-genaseGAPDHPCＲ。、FAM、VICＲOX。2CYP2C19*2、CYP2C19*3CYP2C19*17、CYP2C19gDNAPCＲCYP2C19PCＲ。PCＲ5xPCＲbuffer5μLdNTPmix0.15mmol·L－10.2μmol·L－10.05～0.5μmol·L－1Taq0.02U2μLddH2O25μL。PCＲ95℃5min195℃15s60℃1min40。3CYP2C19*2、*3*17PCＲCYP2C19681G＞A、636G＞A－806C＞T。。2.22CYP2C19*2、CYP2C19*3CYP2C19*17、DNA15ng·μL－10.5ng·μL－1。15ng·μL－1DNA0.5ng·μL－1DNA15ng·μL－1CYP2C19。2.3100gDNA3。2.4SPSS16.0。McNemar－Bowker—3651—ChinJPharmAnal20153592。KappaKappa＞0.75。33.13CYP2C19*2、CYP2C19*3CYP2C19*17。CYP2C19*2、CYP2C19*3CYP2C19*17/0.1/0.2μmol·L－1、0.2/0.3μmol·L－10.3/0.3μmol·L－1。GAPDH0.05μmol·L－1。PCＲCYP2C195xPCＲ、dNTPs、、、Taq23μLDNA2μL。PCＲ60℃62℃。PCＲ95℃5min95℃15s62℃60s40。3.2215ng·μL－1CYP2C19*2、CYP2C19*3CYP2C19*17。3FAMCt＞36CtVICCt≤36FAMVICCt≤36ＲOXCt≤32。1。1Fig．1ＲesultsoftheaccuracytestA．CYP2C19*2homozygousgenotypeCYP2C19*2/*2B．CYP2C19*2heterozygousgenotypeCYP2C19*1/*2C．CYP2C19*3homozygousgenotypeCYP2C19*3/*3D．CYP2C19*3heterozygousgenotypeCYP2C19*1/*3E．CYP2C19*17homozygousgenotypeCYP2C19*17/*17F．CYP2C19*17heterozygousgenotypeCYP2C19*1/*17WP．probesignalforwildgenotypesMP．probesignalformutantgenotypesICON．probesignalforinternalstandard3.320.5ng·μL－13。3FAMVICCt≤36ＲOXCt≤32。1nggDNA。2。—4651—ChinJPharmAnal20153592Fig.2ＲesultsofthesensitivitytestA．CYP2C19*2heterozygousgenotypeCYP2C19*1/*2B．CYP2C19*3heterozygousgenotypeCYP2C19*1/*3C．CYP2C19*17heterozygousgenotypeCYP2C19*1/*17WP．probesignalforwildgenotypesMP．probesignalformutantgenotypesICON．probesignalforinternalstandard3.4215ng·μL－1CYP2C19*1/*13。3FAMCt≤36VICCt＞36Ct。3。3Fig．3ＲesultsofthespecificitytestA．CYP2C19*2wildgenotypesCYP2C19*1/*1B．CYP2C19*3wildgenotypesCYP2C19*1/*1C．CYP2C19*17wildgenotypesCYP2C19*1/*1WP．probesignalforwildgenotypesMP．probesignalformutantgenotypesICON．probesignalforinternalstandard—5651—ChinJPharmAnal20153593.5100。CYP2C1936CYP2C19*1/*236/1009CYP2C19*2/*29/1006CYP2C19*1/*36/1001CYP2C19*3/*31/1002CYP2C19*2/*32/1002CYP2C19*1/*172/1001CYP2C19*17/*171/100。Kappa=1.001。12CYP2C19Tab．1ComparisonofthetwoCYP2C19polymorphismdetectionmethodsPCＲfluorescencePCＲmethodDNAdirectsequencingmethod*1/*1*1/*2*2/*2*1/*3*2/*3*3/*3*1/*17*17/*17total*1/*143000000043*1/*203600000036*2/*2009000009*1/*3000600006*2/*3000020002*3/*3000001001*1/*17000000202*17/*17000000011total4336962121100Kappa=1.004CYP2C191993Wrighton10CYP450、、、。CYP2C19。CYP2C19*17*17/*17CYP2C19*2/*2*3/*3CYP*2/*3。。、、per