Chien-Yu Lin1,2,3, Cheng-Mao Ho2,4, Gevorg Tamamyan5, Shu-Fen Yang2,3, Ching-Tien Peng6,7, Jan-Gowth Chang8,9,10. 1. Graduate Institute of Clinical Medical Sciences, China Medical University, Taichung, Taiwan. 2. Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan. 3. Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan. 4. Department of Nursing, Hungkuang University, Taichung, Taiwan. 5. Department of Pediatric Hemato/Oncology, Complex Clinic of Chemotherapy, Yerevan State Medical University "Muratsan" Hospital, Yerevan, Armenia. 6. Division of Pediatric Hemato/Oncology, China Medical University Children's Hospital, Taichung, Taiwan. pct@mail.cmuh.org.tw. 7. Department of Biotechnology, Asia University, Taichung, Taiwan. pct@mail.cmuh.org.tw. 8. Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan. D6781@mail.cmuh.org.tw. 9. Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan. D6781@mail.cmuh.org.tw. 10. College of Medicine, China Medical University, Taichung, Taiwan. D6781@mail.cmuh.org.tw.
Abstract
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.
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.
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