title: A multiplex snapback primer system for the enrichment and detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia-negative myeloproliferative neoplasms.
abstract: A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.
authors.affiliation: Central Laboratory, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China ; Department of Laboratory Medicine, Huashan Hospital North, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Zhiyuan
authors.identifier: 0000-0002-0466-5996
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Department of Dermatology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
authors.forename: Yunqing
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Central Laboratory, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Xinju
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Central Laboratory, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Kang
authors.affiliation: Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Shibao
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Laboratory Medicine, Huashan Hospital North, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Su
authors.affiliation: Central Laboratory, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China ; Department of Laboratory Medicine, Huashan Hospital North, Shanghai Medical College, Fudan University, Shanghai, China ; Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Ming
authors.identifier: 0000-0002-8796-2653
authors.initials: M
authors.lastname: Guan
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis* / Q000235:genetics*; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D053628:Receptors, Thrombopoietin / Q000235:genetics*
chemical_list: D017931:DNA Primers; D005819:Genetic Markers; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; C507924:JAK2 protein, human; D053614:Janus Kinase 2
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