荧光PCR结合熔解曲线法检测JAK2 V617F突变方法的建立及其方法学验证  

Establishment and analytical validation of JAK2 V617F mutation assay by fluorescence PCR combined with melting curve analysis

作　　者：胡婷婷[1] 陈宇明[1] 张心菊[2] 顾小叶[2] 关明[1] 刘维薇[3] 

机构地区：[1]复旦大学附属华山医院检验科,上海200040 [2]复旦大学附属华山医院中心实验室,上海200040 [3]同济大学附属第十人民医院检验科

出　　处：《中华检验医学杂志》2013年第9期796-800,共5页Chinese Journal of Laboratory Medicine

基　　金：国家临床重点专科资助项目;上海市科学技术委员会基金资助项目（11JC1401800）;复旦大学卓学人才计划（2011年度）资助项目

摘　　要：目的 参考美国病理学家协会（CAP）相关要求,验证本实验室自建的JAK2 V617F突变检测项目的各项性能参数.方法 方法学建立.收集2010年4月至12月复旦大学附属华山医院已确诊的57例BCR/ABL融合基因阴性骨髓增殖性肿瘤患者的外周血和骨髓液标本.采用荧光PCR结合熔解曲线法建立JAK2 V617F突变方法检测标本.在准确度、重复性、分析灵敏度、干扰物质的影响等各方面对检测方法的性能进行评估.准确性使用Kappa检验评估,重复性使用符合率,分析灵敏度使用配对t检验进行统计分析.结果 荧光PCR结合熔解曲线方法（试验方法）与参考方法（测序法）相比有非常好的一致性（Kappa=0.89）；重复性符合率为100％；血脂（＜24 mmol/L）和黄疸（＜450 μmol/L）对本试验无明显干扰；最低检测突变率为5％.结论 基于荧光PCR结合熔解温度反应方法检测JAK2 V617F突变可应用于临床检测.Objective According to the requirements of the College of American Pathologists （CAP）, to validate the laboratory developed test-JAK2 V617F mutation. Methods This is a clinical laboratory diagnosis study. Fifty-seven peripheral blood and bone marrow samples were collected from the BCR/ABL-negative myeloproliferative neoplasms （MPN） patients in Huashan Hospital from Apr 2010 to Dec 2010. JAK2 V617F mutation was detected by fluorescence PCR combined with melting curve analysis. The accuracy, precision, analytical sensitivity, and anti-interference ability of the assay were validated. The Kappa test was used to evaluate accuracy. Coincidence rote was used to evaluate the repeatability. Paired t test was used to evaluate analytical sensitivity. Results There was a close agreement between the reference method（sequencing） and the test method （fluorescence melting curve analysis） （Kappa = 0. 89 ）. The coincidence rate was 100%. The results of the assay was not affected by lipoprotein （ 〈 24 mmol/L ） or bilirubin （ 〈450 panol/L）. The detection limit of the assay was 5%. Conclusion JAK2 V617F mutation assay by fluorescence PCR combined with melting curve analysis could be applied to the clinical laboratory.

关 键 词：JANUS激酶2 聚合酶链反应 突变 

分 类 号：R551.3[医药卫生—血液循环系统疾病]