Simultaneous detection of JAK2 V617F mutation and JAK2 exon12 mutations by multiplex PCR and high-resolution melting curve analysis
10.3760/cma.j.issn.1009-9158.2014.03.008
- VernacularTitle:采用多重PCR结合高分辨熔解曲线法单管同时检测JAK2V617F突变和JAK2外显子12突变
- Author:
Xiao XU
;
Yuming CHEN
;
Zhiyuan WU
;
Xinju ZHANG
;
Tingting HU
;
Jin ZHANG
;
Ming GUAN
- Publication Type:Journal Article
- Keywords:
Janus kinase 2;
Exons;
Mutation;
Multiplex polymerase chain reaction;
High resolution melting;
Polycythemia vera
- From:
Chinese Journal of Laboratory Medicine
2014;37(3):194-197
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a single-tube detecting system for the simultaneous identification of JAK2 V617F and JAK2 exon12 mutations.Methods Genomic DNA of cell line PC-3 was utilized as the wild type control,while genomic DNA of cell line HEL and plasmids with diverse JAK2 exon 12 mutations were used as the positive controls for JAK2 V617F and exon12 mutations.Multiplex PCR was performed to amplify the different amplicons combined with high-resolution melting (HRM) analysis,which established the multiplex detecting system for JAK2 V617F and exon12 mutations.Meanwhile 42 cases of polycythemia vera patients were collected to detect 2 kinds of JAK2 mutations by the above system and routine methods.Results The multiplex JAK2 mutations detecting system was successfully established by multiplex PCR combined with high-resolution melting curve analysis,which could simultaneously detect JAK2 V617F and JAK2 exon12 mutations.The analytical sensitivities of 2 mutations in this system were both up to 5％ and the precision (coefficient of variation) of intra-and inter-assay of the melting temperature (Tm) of 2 amplicons were separately less than 0.01％.37 cases were identified JAK2 V617F mutations from 42 polycythemia vera patients,while 2 JAK2 exon12 mutations cases were found from 5 JAK2 V617F negative patients.Compared with routine methods,the results matched the rate of 100％.Two cases of JAK2 exon 12 mutations were confirmed to the mutation types of H538K539delinsL and F537-I546dul10 + F547L by cloning and sequencing.Conclusions This method can simultaneously detect two kinds of JAK2 mutations in the peripheral blood and will contribute to the molecular diagnosis of myeloproliferative neoplasms,especially polycythemia vera.
