Development and Validation of a Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Combined with Melting Analysis-Assay for Clinical JAK2 V617F Mutation Detection

JAK2 V617F mutation is a molecular marker for myeloproliferative neoplasms (MPNs). As there are no China Food and Drug Administration-approved assays for the detection of JAK2 V617F mutation in China, validation of the analytic performance of this assay is important for the clinical laboratory before its clinical implementation. We have established a method for detecting JAK2 V617F using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR) combined with melting-curve analysis. A total of 202 blood samples and 20 bone marrow aspirates were obtained from MPNs patients at Huashan Hospital, Fudan University. The accuracy, precision, reproducibility, analytical sensitivity, lower limit of detection, analytical specificity, interfering substances, ruggedness, robustness, reportable range and reporting of this assay were validated. There was a close agreement between the reference method (sequencing) and melting-curve analysis (kappa = 0.89). The precision was 100 % and the results of the assay were unaffected by lipoprotein (< 27 mmol/L) or bilirubin (< 450 A mu mol/L). The analytical sensitivity of the JAK2 mutation was 1.25 %. Tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR) combined with melting-curve analysis can be used in the clinical laboratory for detection of the JAK2 V617F mutation.