title: [A novel modi cation of real-time AS-qPCR by using locked nucleic acid-modified oligonucleotide probe as wild type allele amplification blockers for quantitative detection of the JAK2 V617F mutation].
abstract: OBJECTIVE
  To develop a novel real-time PCR for sensitively quantitative detection of JAK2 V617F allele burden in peripheral blood.
  
  
  METHODS
  Based on the real-time allele-specific PCR (AS-qPCR), the locked nucleic acid (LNA)-modified oligonucleotide probe was used for selectively blocking amplification of wild-type alleles in AS-qPCR, and then a novel AS-LNA-qPCR method was established. The percentages of sample JAK2 V617F alleles were directly calculated by its threshold cycle (Ct) values according to the standard curve which generated by JAK2 V617F alleles with its Ct values. We validated intra- and inter-assay variability for quantifying JAK2 V617F. We also assayed 623 apparent healthy donors by our method to validate its clinical application value.
  
  
  RESULTS
  The quantitative lower limit of this method for JAK2 V617F was 0.01%, and the intra- and inter-assay average variability for quantifying percentage of JAK2 V617F in total DNA was 6.3% and 8.6%, respectively. Nineteen JAK2 V617F-positive individuals were identified using AS-LNA-qPCR in blood of 623 apparently healthy donors, and the range of percentages of JAK2 V617F alleles were 0.01%-5.49%.
  
  
  CONCLUSIONS
  The AS-LNA-qPCR with highly sensitive and reproducible quantification of JAK2 V617F mutant burden can be used clinically for diagnosis as well as evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms.
authors.affiliation: Department of Clinical Laboratory, Aerospace Center Hospital, Beijing 100049, China.
authors.forename: Dong-hua
authors.initials: DH
authors.lastname: Shao
authors.forename: Guo-wei
authors.initials: GW
authors.lastname: Liang
authors.forename: Mei-lin
authors.initials: ML
authors.lastname: He
authors.forename: Qing-yun
authors.initials: QY
authors.lastname: Cao
mesh_terms: D000483:Alleles; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D015345:Oligonucleotide Probes; D009841:Oligonucleotides; C477371:locked nucleic acid; C507924:JAK2 protein, human; D053614:Janus Kinase 2