Rapid detection of JAK2 V617F mutation using high-resolution melting analysis with LightScanner platform

Abstract

Background Detection of the JAK2 mutation has recently been included under the essential diagnostic criteria for myeloproliferative neoplasm (MPN). High-resolution melt (HRM) curve analysis, a nongel-based, automated system, is introduced as a means of mutation scanning without the requirement of any post-PCR handling. Methods We studied the sensitivity and reproducibility of LightScanner™ platform in the detection of JAK2 V617F mutation and the availability for diagnostic use in MPN. Results The reproducible sensitivity of HRM analysis with LightScanner™ platform was 5% for the detection of JAK2 V617F mutation. In the test of blind screening of 105 samples (48 Ph− MPN and 57 Ph+ chronic myeloid leukemia), the identical judgement was interpreted by two blinded investigators. HRM analysis of all cases was fully concordant with the results of PCR-RFLP and direct sequencing. Conclusions The HRM method developed here is an extremely sensitive, accurate and reliable technique and allows high-throughput, fast pre-screening to select for sequencing only those specimens that most likely contain mutant JAK2 V617F allele(s).