多重PCR结合毛细管电泳检测骨髓增殖性肿瘤患者JAK2V617F及CALR基因突变  

Detection of JAK2V617F and CALR Gene Mutations by Multiplex of Patients with Myeloproliferative Neoplasms PCR-Capillary Electrophoresis

作　　者：袁建龙[1] 师迎旭[1] 杜华[2,3] 王颖君 赵子玲[1] 李歌[1] 韩艳秋 YUAN Jian-Long;SHI Ying-Xu;DU Hua;WANG Ying-Jun;ZHAO Zi-Ling;LI Ge;HAN Yan-Qiu(Department of Clinical Laboratory Examination,Affiliated Hospital of Inner Mongolia Medical University,2Department of Pathology,Affiliated Hospital of Inner Mongolia Medical University,3Basic Medical College,Inner Mongolia Medical University,Huhhot 010050,Inner Mongolia Autonomous Region,China)

机构地区：[1]内蒙古医科大学附属医院检验科,内蒙古呼和浩特010050 [2]内蒙古医科大学附属医院病理科,内蒙古呼和浩特010050 [3]内蒙古医科大学基础医学院,内蒙古呼和浩特010050

出　　处：《中国实验血液学杂志》2020年第6期1998-2003,共6页Journal of Experimental Hematology

基　　金：内蒙古医科大学科技百万工程(YKD2015KJBW010);内蒙古自然科学基金(2018BS03013)。

摘　　要：目的:应用多重PCR与毛细管DNA电泳法检测骨髓增殖性肿瘤(MPN)主要致病基因中的JAK2V617F及CALR第九外显子突变,并对该方法的检测性能进行评价。方法:同时设计特异性的JAK2617F等位基因突变PCR引物以及CALR基因第九外显子扩增引物,对引物进行Cy5荧光标记,所有引物在同一PCR反应管中进行扩增,并将PCR产物进行毛细管电泳分析,同时验证该方法的检测限、灵敏度,并与商品化试剂盒进行对比。结果:多重PCR与毛细管电泳技术结合可在一个PCR反应中同时检测JAK2V617F与CALR基因突变,可以在0.01 ng的基因组DNA中检测出JAK2V617F突变,可在0.1 ng的基因组DNA检测出JAK2V617F与CALR双阳性突变,至少可检测出0.1%的JAK2V617F阳性突变,该方法与商品化诊断试剂盒进行对比结果相一致。结论:基于多重PCR与毛细管电泳技术,可在外周血中同时检测JAK2V617F与CLAR基因第九外显子突变,该方法为MPN的诊断提供了新的分子检测手段。Objective:To evaluate the proformance of multiplex PCR and capillary electrophoresis(MPCE)in the detection of JAK2V617F and CALR mutation in myeloproliferative neoplasms(MPN).Methods:The specificity primers of JAK2617F gene mutation and the primers of CALR gene were designed at the same time.The JAK2V617F and CALR gene primers were labeled with Cy5 fluorescence,all the primers were mixed in one tube for multiplex PCR and the PCR prodcuts were analysised by capillary electrophoresis.Then detection limit and sensitivity of MPCE were evaluated,and compared with comercial diagnostic kit.Results:JAK2V617F and CALR gene mutations could be detect by MPCE in one PCR test.JAK2V617F mutation could be detected at 0.01 ng genomic DNA,double positive JAK2V617F and CLAR gene mutations could be detected at 0.1 ng genomic DNA,at least 0.1%JAK2V617F positive mutation could be detected.The consistency between MPCE and commercial diagnostic gene mutation kit was 100%.Conclusion:It is developed that a new gene mutation detection method of JAK2 V617F and CLAR gene based on MPCE in our experiment and it can be used as a new reagent for molecular diagnosis of MPN patients.

关 键 词：多重PCR 毛细管电泳 骨髓增殖性肿瘤 JAK2V617F CALR 

分 类 号：R733.[医药卫生—肿瘤]