TaqMan探针结合COLD-PCR原理定量检测JAK2V617F突变率研究

摘要

目的 建立在外周血基因组DNA中定量检测JAK2 V617F突变率的方法.方法 应用TaqMan/MGB探针技术,结合较低变性温度下复合扩增聚合酶链反应(COLD-PCR)基因扩增原理,采用Real-Time PCR分析系统,通过JAK2 V617F突变率和其循环阈值(Ct值)的标准曲线,建立TaqMan-COLD-PCR测定方法,并对9例骨髓增殖性疾病(MPD)患者外周血基因组DNA中JAK2 V617F突变率进行检测,探讨其应用价值.结果 建立的TaqMan-COLD-PCR方法 检测JAK2V617F突变率的检测下限为0.1%,100%-0.1%的JAK2 V617F突变率与其测定的Ct值间呈明显的线性关系.对9例MPD患者外周血基因组DNA中JAK2 V617F突变率检测显示,其中6例患者的JAK2 V617F突变阳性,突变率范围为18.5%-50.9%,经测序确认全部符合.结论 TaqMan-COLD-PCR方法 可高灵敏度、定量检测外周血中JAK2 V617F突变率.该方法 将明显提高MPD疾病的诊断灵敏度,并有助于疾病治疗过程中治疗效果的监测.%Objective To develop a real-time PCR test for quantitatively detecting mutation rate of JAK2 V617F in blood sample. Methods Combining exemplification at Lower Dcnaturation Tcmpcraturc-PCR(COLD-PCR) with Taq-Man genotyping method in a real-time format,the TaqMan-COLD-PCR method by standard curve of mutation rate and threshold cycle values (Ct) for quantitatively detect mutation rate of JAK2 V617F in blood sample was established. In order to investigate clinical application value of method we developed, 9 of mycloprolifcrativc diseases (MPD) which include 8 of polycythcmia vcra (PV) and 1 of primary myclofibrosis (MF) cases were tested by the method. Results The developed method detect sensitivity was 0.1% for JAK2 V617F mutation,and the linear relationship between 100% - 0. 1 % mutation rate of JAK2 V617F and its Ct values were also observed by TaqMan-COLD-PCR method. There were 6 JAK2 V617F mutation cases detected in the 9 MPD blood samples, and the range of mutation rate was 18. 5%- 50. 9% in 6 JAK2 V617F positive samples. The tested results between TaqMan-COLD-PCR method and DNA Sequencing were consistent completely in 9 MPD samples. Conclusion The TaqMan-COLD-PCR method we developed can quantitatively screen JAK2 mutations with high sensitivity in blood samples, by which the diagnostic sensitivity and therapeutic monitoring of MPD patients could be highly improved.