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

Preliminary Study on Simultaneous Detection of JAK2,MPL and Calreticulin Mutations in Patients with Myeloproliferative Neoplasms by Multiplex PCR High Resolution Melting Analysis

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 kinase2,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(polymerase chain 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,JAK2V617F和外显子12的引物需重新设计。

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.