本研究建立在外周血基因组DNA中定量检测JAK2V617F突变率的荧光定量PCR检测方法并探讨其临床应用价值.在实时荧光定量PCR分析系统中,应用等位基因突变引物和等位基因野生TaqMan-MGB探针共同抑制JAK2 V617F野生等位基因扩增,从而特异性地扩增JAK2V617F突变等位基因.通过测定不同JAK2V617F突变率标准品及其循环阈值(Ct值),建立JAK2 V617F突变率定量测定方法,并对89例表观健康人外周血基因组DNA中JAK2V617F突变率进行检测.结果表明,建立的方法定量检测JAK2V617F突变率的下限为0.1％,检测JAK2V617F突变率的批内、批间平均变异率分别为4.1％和6.1％.对89例表观健康人外周血基因组DNA中JAK2V617F突变率检测显示,其中2例为JAK2V617F阳性,突变率分别为0.64％和0.98％.结论:建立的JAK2V617F突变率定量检测方法具有检测灵敏度高和重复性好的特性,适合骨髓增殖性疾病的诊断及疾病进程和疗效监测.本方法检测原理可用于各种单碱基突变率的检测,具有广泛的应用前景.%This study was purposed to develop a real-time PCR assay for sensitive quantification of JAK2V617F allele burden in peripheral blood and to evaluate the clinical value of this method. Both allele-specific mutant reverse primer and wild-type TaqMan-MGB probe were used for dual-inhibiting amplification of wild-type alleles in a real-time PCR, and then the JAK2V617F mutant alleles were amplified specially. The standard curve for quantification of JAK2V617F was established by percentages of JAK2V617F alleles with threshold cycle (Ct) values in a real-time PCR. Furthermore, 89 apparent healthy donors were tested by this method. The results showed that the quantitative lower limit of this method for JAK2V617F was 0.1% , and the intra- and inter-assay average variability for quantifying percentage of JAK2V617F in total DNA was 4.1% and 6.1% , respectively. Two JAK2V617F-positive individuals were identified (the percentage of JAK2V617F alleles were 0. 64% and 0. 98% , respectively) using this method in blood from 89 apparently healthy donors. It is concluded that the developed method with highly sensitive and reproducible quantification of JAK2V617F mutant burden can be used clinically for diagnosis and evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms. Moreover, this technique can be also used for quantitative detection of variety of single nucleotide mutation.
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