Development and Inter-Laboratory Validation of Unlabeled Probe Melting Curve Analysis for Detection of

Background: JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Methodology/Principal Findings: Asymmetric PCR for detection of JAK2 V617F with a 39-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-GeneH Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCyclerH 480, Applied Biosystems ABIH 7500 and Eppendorf MastercyclerH ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3 % mutation load detecting sensitivity. At level of 5 % mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55 % and 1.72%/1.29 % respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments