骨髓增生疾病JAK2 V617F突变快速检测体系的建立  

Establishment of a system for rapid detection of JAK2 V617F mutation in myeloproliferative diseases

作　　者：张强[1] 张艺佳 徐湘民 周万军 范歆[1] Zhang Qiang;Zhang Yijia;Xu Xiangmin;Zhou Wanjun;Fan Xin(Central Laboratory for Inborn Error of Metabolism,Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region,Nanning,Guangxi 530003,China;Department of Medical Genetics,School of Basic Medical Sciences,Southern Medical University,Guangzhou,Guangdong 510515,China)

机构地区：[1]广西壮族自治区妇幼保健院遗传代谢中心实验室,南宁530000 [2]南方医科大学基础医学院医学遗传学教研室,广州510515

出　　处：《中华医学遗传学杂志》2019年第10期980-984,共5页Chinese Journal of Medical Genetics

摘　　要：目的建立一种骨髓增生性疾病JAK2 V617F突变快速检测体系,并调查中国正常人群中JAK2 V617F突变发生率.方法根据JAK2基因V617F位点DNA参考序列,分别设计靶序列扩增特异性引物、突变和野生型特异性TagMan探针,建立并优化基于实时定量荧光PCR技术的检测体系并对体系的灵敏度、特异性,重复性进行评价.同时,采用大引物定点诱变和直接克隆技术,构建相应的阴阳性质粒DNA,以形成包含检测方法与质控品的完整应用体系.结果建立了骨髓增生性疾病JAK2 V617F突变快速检测体系,此体系与传统Sanger测序比较,灵敏度和特异性均为100%,批内变异系数为0.006.同时,对1000份正常样本进行检测,检出JAK2 V617F突变1例,人群携带率1‰.结论本研究建立的检测体系操作简单、快速准确,低成本高通量,可应用于常规临床检测.JAK2 V617F突变是一种罕见突变,但在骨髓增生性疾病患者中应作为首先检测位点.Objective To develop a system for rapid detection of JAK2 V617F mutation among patients with myeloproliferative diseases.Methods Specific primers and TagMan probes were designed for the mutant and wild type alleles based on the principle of real-time PCR.A complete system including the method for detection and product for quality control were established through the evaluation of sensitivity and accuracy of the method,double-blind trial,and preparation of negative and positive controls through site-directed mutagenesis and molecular cloning.Results A system for rapid detection of the JAK V617F mutation has been developed.Compared with Sanger sequencing,the sensitivity and specificity of the method have both reached 100%.Meanwhile,1000 normal samples and 1 case with the JAK2 V617F mutation were detected,which gave a population rate of 1‰.Conclusion The system was fast,accurate,cheap,high throughput,and easy to use.It can be utilized as a routine test.Although the JAK2 V617F mutation is rare in the population,it should be screened among myeloproliferative neoplasm patients.

关 键 词：骨髓增生性疾病 基因突变 TagMan探针 人群携带率 

分 类 号：R5[医药卫生—内科学]