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时间:2020-06-04
《高分辨熔解曲线分析法检测急性髓细胞白血病IDH2基因突变.pdf》由会员上传分享,免费在线阅读,更多相关内容在应用文档-天天文库。
1、·664·临床检验杂志2012年9月第30卷第9期ChinJClinLabSci,Sep.2012,Vo1.30,No。9文章编号:1001-764X(2012)09-664-04·临床检验技术研究·高分辨熔解曲线分析法检测急性髓细胞白血病IDH2基因突变薛娣,邵睿,林江,肖高飞,钱军,姚冬明,柴海彦。,李云,杨静,王翠竹,陈星星。(江苏大学附属人民医院a.病理科,b.实验中心,e.血液科,江苏镇江212002)摘要:目的建立高分辨熔解曲线分析(HRMA)技术检测急性髓细胞白血病(AML)中可溶性异柠檬酸脱氢酶2(IDH2)基因突变。方法针
2、对IDH2突变位点设计PCR扩增引物,建立带有温度内标校准品的PCR扩增体系,对98例AML患者标本进行PCR扩增,用HRMA技术对PCR产物进行突变分析,并通过DNA测序技术对HRMA结果进行验证。结果对98例AML样本的PCR产物进行HRMA检测,发现5例存在异常熔解曲线,对此5例标本及随机选择的HRMA显示为野生型的35例IDH2标本进行DNA测序验证,结果表明,存在异常熔解曲线的5例标本为4例R140Q突变和1例R172K突变,其余35例经DNA测序证实为野生型IDH2;对不同浓度梯度的R140Q质粒HRMA检测结果表明其灵敏度为2
3、%。结论成功建立HRMA技术检测IDH2突变,其可用于AML标本的临床检测。关键词:高分辨熔解曲线分析;可溶性异柠檬酸脱氢酶2;基因突变;急性髓细胞白血病中图分类号:R733.71文献标志码:AHigh-resolutionmeltingcurveanalysisfordetectionofisocitratedehydrogenase2mutationsinacutemyeloidleukemiaXUEDi,SHAORui,LINJiang。,XIAOGao-fei,QIANJun,YAODong—ming。,CHAIHai—yan,LI
4、Yun,YANGJing。,WANGCui—zhu,CHEN一xing(1.DepartmentofPathology;2.LaboratoryCenter;3.DepartmentofHematology,AfiliatedPeopleSHospitalofJiangsuUniversity,Zhenjiang212002,Jiangsu,China)Abstract:ObjectiveToestablishhigh—resolutionmeltingcurveanalysis(HRMA)techniquefordetectingisoc
5、itratedehydrogenase2(IDH2)mutationsinacutemyeloidleukemia(AML).MethodsTheprimersforIDH2mutationsweredesignedandPCRsystemwithinternaltemperaturecalibratorswasestablished.Thebloodsamplesfrom98AMLpatientswereamplifiedandanalyzedbyHRMA.Di—rectDNAsequencingwasperformedtoconfirm
6、theresultsofHRMA.ResultsFor5ofthe98AMLsamplesabnormalmeltingCurveswereidentifiedinHRMA.The5sampleswithabnormalmeltingcurvesand35randomlyselectedsampleswithwild—typeDNAdetectedbyHRMAwerecheckedbysequencing,andR140Qmutationwasfoundin4samplesandR172Kmutationwasin1sample.The35
7、sampleswerealsoconfirmedaswild—typebyDNAsequencing.ThesensitivityoftwopercentwasdeterminedbyHRMAforplasmidDNAwithdifferentconcentrationsofR140Qmutant.ConclusionTherapid.easyandsensitiveI-IRMAmethodwasestablishedandmaybeusedforhigh—throughputscreeningofⅡ)mutationsinAMLpatie
8、nts.Keywords:high.resolutionmeltingcurveanalysis;isocitratedehydrogenase2;genemutation;ac
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