背景:具有临床意义的因子VLeiden(FVL)点突变(1691G/A)导致用Gln(谷氨酰胺)替换Arg,防止活化的蛋白C使因子V失活,导致凝血过程延长。具有因子VLeiden突变的个体静脉血栓形成的风险增加。这项研究的目的是比较未标记的探针高分辨率熔解分析(HRMA)测定因子VLeiden突变与TaqMan水解测定(荧光5'核酸酶PCR水解测定)。HRMA是PCR后,同质,用于检测序列变异的闭管系统。PCR后,将扩增子逐渐加热,直到达到解链温度,并且荧光染料与扩增子不结合并表现出低荧光。产生熔解曲线分析,其是特定序列变体的特征。因此,HRMA允许基于其解链速率的差异来比较遗传序列中的一个碱基变化。
方法:将血液样品收集在EDTA管中,并使用RocheMagNaPure提取DNA。HRMA和TaqMan的反应均在3个对照上进行(1691G/G,1691G/A,和1691G/G和G/A)和20个样品。
结果:从Coriell购买的3个参考对照的基因型(F51691G/G,FVL1691G/A,和杂合子1691G/G和G/A)均通过HRMA和TaqManFVL测定得到证实。通过HRMA和TaqMan测定确认所有20个样品为F51691G/G。
结论:将未标记的探针HRMAFVL测定的结果与实时TaqMan探针终点基因分型测定的结果进行比较,两种测定均具有100%的灵敏度和100%的特异性。
BACKGROUND: The clinically significant Factor V Leiden (FVL) point mutation (1691 G/A) causes replacement of Arg with Gln (glutamine), preventing activated protein C from inactivating Factor V leading to a lengthened clotting process. Individuals with the Factor V Leiden mutations have an increased risk for venous thrombosis. The aim of this study is to compare an unlabeled probe high-resolution melting analysis (HRMA) assay for Factor V Leiden mutation to a TaqMan hydrolysis assay (fluorogenic 5\' nuclease PCR hydrolysis assay). HRMA is a post-PCR, homogenous, closed-tube system for the detection of sequence variants. Post-PCR, the amplicons are heated gradually until the melting temperature is reached and the fluorescent dye unbinds from the amplicon and exhibits low fluorescence. A melt-curve analysis is generated that is characteristic of a particular sequence variant. Therefore, HRMA allows for comparison of one base changes in genetic sequences based on their differences in melting rate.
METHODS: Blood samples were collected in EDTA tubes and DNA extracted using the Roche MagNaPure. Reactions of both HRMA and TaqMan were carried out on 3 controls (1691 G/G, 1691 G/A, and 1691 G/G and G/A) and 20 samples.
RESULTS: The genotypes for 3 reference controls purchased from Coriell (F5 1691 G/G, FVL 1691 G/A, and Heterozygote 1691 G/G and G/A) were confirmed by both the HRMA and TaqMan FVL assays. All 20 samples were confirmed to be F5 1691 G/G by both HRMA and TaqMan assays.
CONCLUSIONS: Comparing the results of the unlabeled probe HRMA FVL assay with a real-time TaqMan probe end point genotyping assay resulted in 100% sensitivity and 100% specificity for both assays.