Open AccessValidating the Sensitivity of High‐Resolution Melting Analysis for JAK2 V617F Mutation in the Clinical Setting
Author(s) -
Lin ChienYu,
Ho ChengMao,
Tamamyan Gevorg,
Yang ShuFen,
Peng ChingTien,
Chang JanGowth
Publication year - 2016
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.21945
Subject(s) - high resolution melt , janus kinase 2 , mutation testing , melting curve analysis , jak2 v617f , mutation , polymerase chain reaction , microbiology and biotechnology , myeloproliferative neoplasm , biology , genetics , kinase , immunology , gene , myelofibrosis , bone marrow
Background Janus kinase 2 ( JAK2 ) plays an important role in normal hematopoietic growth factor signaling. The detection of the JAK2 V617F mutation (c.1849GNT, GTC → TTC) is crucial for the diagnosis of myeloproliferative neoplasm (MPN) and has become the essential criteria for diagnosis of MPN by the WHO. High‐resolution melt (HRM) curve analysis is a nongel‐based, closed‐tube method, in which PCR amplification and subsequent analysis are sequentially performed in the well, making it more convenient than other scanning methodologies. Methods We evaluated JAK2 V617F mutation by HRM. Twenty‐nine patients diagnosed with MPN were examined. We studied the analytical sensitivity of the HRM analysis using real‐time polymerase chain reaction (PCR) for identifying the JAK2 V617F mutation. Additionally, the sensitivity of HRM analysis and allele‐specific PCR (AS‐PCR) assay was compared. Results The JAK2 V617F mutation was successfully discriminated at an abundance of 6% or above in HRM analysis. Both HRM analysis and AS‐PCR showed 100% accuracy with detection limits of 6% and 2.5%, respectively. Conclusion HRM analysis is a fast, simple, reliable, and nonexpensive method for the detection of the JAK2 V617F mutation. However, more validation of the detection limits of HRM analysis should be performed before declaration of the analytic sensitivity of the method.