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	双重PCR检测急性髓系白血病FLT3-ITD和NPM1基因突变; Simultaneous Detection of FLT3-ITD and NPM1Gene Mutations in Acute Myeloid Leukemia by Double PCR
	张泽川 ; 鹿全意 ; 赵江宁 ; 陈亚玫 ; 李志鹏
	2011
关键词	白血病 NPM1基因 FLT3基因 内部串联重复 毛细管电泳 leukemia NPM1 FLT3 internal tandem duplication capillary electrophoresis
英文摘要	本研究旨在建立一种同时筛查flT3-ITd突变和nPM1突变的检测方法。设计2对引物,分别扩增nPM1基因的外显子12和flT3基因的外显子14、内含子14、外显子15,以覆盖几乎所有已知突变位点。对双重PCr体系的反应程序和引物浓度比例进行优化,将双重PCr产物通过毛细管电泳分离,根据野生型产物和突变型产物的大小差异来判断突变存在与否并利用产物的峰面积对突变比例进行定量,并对突变阳性标本进行测序验证。结果表明,在93例标本中nPM1突变者17例(18.5%),flT3-ITd突变者15例(16.3%),nPM1突变和flT3-ITd突变双阳性6例。17例nPM1突变中7例M2、4例M4、5例M5、1例M6,其中10例男性、7例女性;有15例为A型,1例为b型,1例为nM型;有1例CMl急变为AMl的标本中带有nPM1基因A型突变。15例flT3-ITd阳性中1例M1、8例M2、2例M3、1例M4、3例M5,其中5例男性、10例女性。扩增产物测序结果进一步证明了该检测体系的准确性和可靠性。结论 :建立了同时筛查flT3-ITd突变和nPM1突变的检测体系,该体系以基因组dnA为模板,具有方便、快捷、准确、可定量的优点。; Objective of this study was to establish a method for simultaneous detection of FLT3/ITD and NPM1 gene mutations in AML.A double PCR was firstly designed and optimized to amplify both exon 12 of NPM1 and exon 14-intron 14-exon 15 of FLT3,with the aim of detecting almost all reported mutations.After optimization,a touchdown PCR was chosen for the multiplex PCR procedure,with the primer concentrations of NPM1 and FLT3-ITD being 200 nmol/L and 152 nmol/L respectively.The PCR amplicons were separated by capillary electrophoresis and the presence of mutants was recognized by the size difference between the mutants and wild-type products.The areas of mutant peak and wild-type peak were used to calculate the mutant/wild-type ratio.All the positive mutated samples were confirmed by sequencing.The results showed that 17 patients with NPM1 mutation,15 patients with FLT3-ITD mutation,6 patients with both NPM1 and FLT3-ITD mutationts were found among 93 patents.7 patients with M2,4 patients with M4,5 patients with M5 and 1 patients with M6 were found out of 17 patients with NPM1 mutation,in which 10 patients were male and 7 patients were female,15 patients were with type A,1 patients was with type B and 1 patients was with type Nm,strinkingly 1 CML patient in blast crisis was found to carry a type A mutation.Among 15 patients with FLT3-ITD mutation 1 patient with M1,8 patients with M2,2 patients with M2,2 patients with M3,1 patient with M4,3 patients with M5 were found,in which 5 patients were male and 10 patients were lemale.Sequencing results further confirmed the accuracy and reliability of this method.It is concluded that a novel method with the ability to detect both FLT3-ITD and NPM1 mutations has been developed when genomic DNA was templated.This method is fast,easy,accurate and capable to calculate the mutant/wild-type ratio.
语种	zh_CN
内容类型	期刊论文
源URL	[http://dspace.xmu.edu.cn/handle/2288/119100]
专题	生命科学－已发表论文
推荐引用方式 GB/T 7714	张泽川,鹿全意,赵江宁,等. 双重PCR检测急性髓系白血病FLT3-ITD和NPM1基因突变, Simultaneous Detection of FLT3-ITD and NPM1Gene Mutations in Acute Myeloid Leukemia by Double PCR[J],2011.
APA	张泽川,鹿全意,赵江宁,陈亚玫,&李志鹏.(2011).双重PCR检测急性髓系白血病FLT3-ITD和NPM1基因突变..
MLA	张泽川,et al."双重PCR检测急性髓系白血病FLT3-ITD和NPM1基因突变".(2011).

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