[1]杨晓慧a,许 蕾b,晏耀明a,等.多重PCR 高分辨熔解分析同时检测骨髓增殖性肿瘤JAK2，MPL 及CALR 基因突变的初步研究[J].现代检验医学杂志,2023,38(04):63-66+71.[doi:10.3969/j.issn.1671-7414.2023.04.011]
　YANG Xiaohuia,XU Leib,et al.Preliminary Study on Simultaneous Detection of JAK2, MPL and Calreticulin Mutations in Patients with Myeloproliferative Neoplasms by Multiplex PCR High Resolution Melting Analysis[J].Journal of Modern Laboratory Medicine,2023,38(04):63-66+71.[doi:10.3969/j.issn.1671-7414.2023.04.011]
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多重PCR 高分辨熔解分析同时检测骨髓增殖性肿瘤JAK2，MPL 及CALR 基因突变的初步研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年04期
页码:
63-66+71
栏目:
论著
出版日期:
2023-07-15

文章信息/Info

Title:
Preliminary Study on Simultaneous Detection of JAK2, MPL and Calreticulin Mutations in Patients with Myeloproliferative Neoplasms by Multiplex PCR High Resolution Melting Analysis
文章编号:
1671-7414（2023）04-063-05
作者:
杨晓慧1a; 2; 许 蕾1b; 晏耀明1a; 李建新1a
（1. 北京大学深圳医院a. 检验科；b. 血液科，广东深圳 518036；2. 深圳大学总医院检验科，广东深圳 518055）
Author(s):
YANG Xiaohui1a;  2; XU Lei1b; YAN Yaoming1a; LI Jianxin1a
（1a. Department of Clinical Laboratory; 1b. Department of Hematology, Peking University Shenzhen Hospital, Guangdong Shenzhen 518036, China; 2. Department of Clinical Laboratory, Shenzhen University Genaral Hospital, Guangdong Shenzhen 518055, China）
关键词:
骨髓增殖性肿瘤; Janus 激酶2; 骨髓增殖性白血病; 钙网素; 多重聚合酶链式反应; 高分辨熔解分析
分类号:
R551.3；Q503；Q786
DOI:
10.3969/j.issn.1671-7414.2023.04.011
文献标志码:
A
摘要:
目的　建立同时检测经典骨髓增殖性肿瘤患者中常见的Janus 激酶2（Janus kinase 2, JAK2）、钙网素（calreticulin,CALR）和骨髓增殖性白血病病毒（myeloproliferative leukemia virus，MPL）基因突变的多重聚合酶链式反应（polymerasechain reaction，PCR）- 高分辨熔解分析（high resolution melting analysis，HRM）体系。方法　先以各个合成的突变等位基因片段为模板分别进行PCR 和HRM 分析，优化PCR 体系后，在单管中进行多重PCR 扩增和HRM 分析，同时检测上述三个基因的8 种常见突变。结果　在适宜的条件下，上述三个基因的野生型和突变型均能扩增出各自的目的条带和收集到可区分的熔解峰，野生型与突变型的熔解温度差为0.3℃～ 2.0℃。多重PCR-HRM 体系中，除JAK2 外显子12 相关片段，其他基因均可有效扩增，但JAK2 V617F 突变型的扩增效率明显低于MPL 及CALR 基因。结论　 目前该多重PCR-HRM 检测体系可鉴别MPL 和CALR 基因常见突变，但JAK2 V617F 突变型由于扩增效率低导致突变峰与野生峰不能明显区分，JAK2 V617F 和外显子12 的引物需重新设计。
Abstract:
Objective　To establish a multiplex polymerase chain reaction(PCR)-high resolution melting analysis (HRM) for simultaneous detection of JAK2, MPL and CALR mutations in philadelphia chromosome negative patients with myeloproliferative neoplasms (MPN). Methods　Firstly, PCR and HRM analysis were performed by using individual synthetic mutant allele fragment as template respectively. Then, multiplex PCR and HRM analysis were carried out in single tube to detect 8 common mutations in the above-mentioned three genes simultaneously after the primers and PCR system were optimized. Results　Under suitable conditions, the wild and mutant alleles of above three genes could be amplified individually, and distinguishable melting peaks could be collected. The melting temperature differences between the wild and the mutant amplicons were 0.3℃～ 2.0℃ . In the multiplex PCR-HRM system, except JAK2 exon 12, other genes could be amplified simultaneously, but the amplification efficiency of JAK2 V617F mutant was significantly lower than that of MPL and CALR. Conclusion　At present, the multiplex PCR-HRM detection system could effectively identify common mutations in MPL and CALR. The JAK2 V617F mutant in the multiplex system could not be clearly differentiated due to poor amplification efficiency. The primers for JAK2 V617F and exon 12 mutations need to be redesigned.

参考文献/References:

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备注/Memo

备注/Memo:
基金项目： 深圳市科技创新委基础研究资助项目（JCYJ20170307111612356）：一管法多重PCR- 高分辨熔解曲线同时检测JAK2，MPL和CALR 突变。
作者简介：杨晓慧（1995-），女，本科，技师，从事分子生物学实验诊断工作，E-mail：lucyyxh913@163.com。
通讯作者：李建新（1968-），男，博士，主任技师，从事血液病实验诊断工作，E-mail：ljxdoctor@126.com。
更新日期/Last Update: 2023-07-15