title: 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.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  There was a close agreement between the reference method (sequencing) and melting-curve analysis (κ = 0.89). The precision was 100 % and the results of the assay were unaffected by lipoprotein (<27 mmol/L) or bilirubin (<450 µmol/L). The analytical sensitivity of the JAK2 mutation was 1.25 %.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Laboratory Medicine, Shanghai Tenth People's Hospital, Tongji University, 301 Yanchang Road, Shanghai, 200072, People's Republic of China, huashanvivian@126.com.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Liu
authors.forename: Tingting
authors.initials: T
authors.lastname: Hu
authors.forename: Yuming
authors.initials: Y
authors.lastname: Chen
authors.forename: Xinju
authors.initials: X
authors.lastname: Zhang
authors.forename: Xiaoye
authors.initials: X
authors.lastname: Gu
authors.forename: Ming
authors.initials: M
authors.lastname: Guan
mesh_terms: D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: C507924:JAK2 protein, human; D053614:Janus Kinase 2