Abstract

To develop a novel platform for detection of the JAK2V617F mutation in patients with myeloproliferative disorders (MPD) by real-time quantitative PCR. TaqMan-MGB probe was constructed. Peripheral blood samples were collected from 374 MPD patients, 76 with polycythemia vera (PV), 38 with chronic myelogenous leukemia (CML), and 115 with essential thrombocythemia (ET), and 19 with idiopathic myelofibrosis (IMF). Peripheral blood samples from 65 patients with acute myelogenous leukemia (AML), 30 patients with acute lymphoblastic leukemia, 8 patients with chronic lymphoblastic leukemia, and 7 patients with non-Hodgkin's lymphoma and 16 cases of normal donor bone marrow were used as controls. Genomic DNA or RNA was extracted and reversely transcrtibed into cDNA. TaqMan-MGB probe was used to detect the JAK2V617F mutant in MPD. Furthermore, 168 specimens underwent allele specific PCR and 8 specimens underwent sequencing. This method was used on both DNA and cDNA specimens from 38 MPD patients simultaneously so as to test the consistency. The JAK2V617F mutation rates of the PV, ET, and IMF patients were 53 (70%), 59 (51%), (58%) respectively. JAK2V617F mutation was found in only one of the 65 AML patients and was not identified in other control specimens. Both the results of allele specific PCR and of sequencing were consistent with the result of TaqMan-MGB probe method. JAK2V617F mutation is widespread in Chinese MPD patients. Real-time quantitative PCR with TaqMan MGB probe can be used for rapid and accurate detection of the JAK2V617F mutation.

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