高分辨率熔解曲线法检测JAK2基因V617F突变及其应用

High resolution melting analysis for rapid and effective detection of JAK2 V617F mutation in clinical samples

目的探讨实时定量PCR方法和高分辨率熔解曲线法(HRM)在检测JAK2基因V617F突变中的应用。方法以JAK2基因V617F发生纯合突变的人红白血病细胞株(HEL)及不含JAK2基因V617F突变的人白血病细胞株(HL60)为阳性对照和阴性对照,优化HRM法检测条件,分别以优化的HRM法和直接测序法对63例临床疑似骨髓增殖性肿瘤(MPN)患者DNA标本进行JAK2基因V617F突变检测,评价2种检测方法结果的一致性。结果 HRM法可检出系列混合样本中5%的突变型等位基因突变,且重复性较高。以测序法为金标准,HRM法的敏感性和特异性均为100%,两种方法结果一致。结论 HRM法能够在单一闭管体系中实现对JAK2基因V617F突变的检测,具有较高的敏感性和特异性,可用于JAK2基因V617F突变的临床检测。

Objectives To explore the application of real time PCR technique and high resolution melting（HRM） analysis in the detection of JAK2 V617F mutation for clinical samples.Methods Two different cell lines（HEL and HL60） were used to validate the capacity of HRM method for detection of JAK2 V617F mutation.The DNA samples from 63 patients clinically suspected with myeloproliferative neoplasm（MPN） were detected by HRM method.The results of HRM analysis were compared with DNA direct sequencing.Results Following PCR amplification,as low as 5% of JAK2 V617F mutation was successfully detected in the suspected MPN patients with MPN using the HRM analysis.High repetition was demonstrated in HRM analysis.The comparability of 100% with the direct sequencing was shown.Conclusion JAK2 V617F mutation could be detected with HRM analysis by performing real time PCR in the same tube on LightCycler 480 analyzer.The method was proven to be sensitive and specific for clinical samples.