CAJ | 학술논문

目的建立能单管同时检测JAK2 V617F突变和外显子12突变的方法.方法以PC-3细胞株DNA为野生型对照,分别以HEL细胞株DNA和含有JAK2外显子12突变的质粒作为JAK2V617F突变和外显子12突变的阳性对照,采用多重PCR扩增不同DNA片段,并结合高分辨熔解曲线法建立检测JAK2V617F和外显子12突变的联合检测体系.同时选取42例真性红细胞增多症患者,分别采用上述方法和常规方法在外周血中检测JAK2的2种突变.结果采用多重PCR结合高分辨熔解曲线法成功建立了JAK2突变联合检测体系,能够同时检测JAK2V617F突变和外显子12突变,2种突变的检测灵敏度皆可达5％,2种扩增片段的溶解温度(Tm)的批间及批内变异系数都小于0.01％.42例真性红细胞增多症患者中,发现37例携有JAK2V617F突变,在5例JAK2V617F突变阴性真性红细胞增多症病例中检出2例JAK2外显子12突变.与常规方法相比,结果符合率达到100％.2例JAK2外显子12突变经克隆测序确认突变类型为H538K539delinsL和F537-I546dul10+F547L.结论该方法可同时在外周血中检测出JAK2的2种突变,将有助于对骨髓增殖性肿瘤尤其是真性红细胞增多症的分子诊断.
목적 건립능단관동시검측JAK2 V617F돌변화외현자12돌변적방법.방법 이PC-3세포주DNA위야생형대조,분별이HEL세포주DNA화함유JAK2외현자12돌변적질립작위JAK2V617F돌변화외현자12돌변적양성대조,채용다중PCR확증불동DNA편단,병결합고분변용해곡선법건립검측JAK2V617F화외현자12돌변적연합검측체계.동시선취42례진성홍세포증다증환자,분별채용상술방법화상규방법재외주혈중검측JAK2적2충돌변.결과 채용다중PCR결합고분변용해곡선법성공건립료JAK2돌변연합검측체계,능구동시검측JAK2V617F돌변화외현자12돌변,2충돌변적검측령민도개가체5％,2충확증편단적용해온도(Tm)적비간급비내변이계수도소우0.01％.42례진성홍세포증다증환자중,발현37례휴유JAK2V617F돌변,재5례JAK2V617F돌변음성진성홍세포증다증병례중검출2례JAK2외현자12돌변.여상규방법상비,결과부합솔체도100％.2례JAK2외현자12돌변경극륭측서학인돌변류형위H538K539delinsL화F537-I546dul10+F547L.결론 해방법가동시재외주혈중검측출JAK2적2충돌변,장유조우대골수증식성종류우기시진성홍세포증다증적분자진단.
Objective To establish a single-tube detecting system for the simultaneous identification of JAK2 V617F and JAK2 exon12 mutations.Methods Genomic DNA of cell line PC-3 was utilized as the wild type control,while genomic DNA of cell line HEL and plasmids with diverse JAK2 exon 12 mutations were used as the positive controls for JAK2 V617F and exon12 mutations.Multiplex PCR was performed to amplify the different amplicons combined with high-resolution melting (HRM) analysis,which established the multiplex detecting system for JAK2 V617F and exon12 mutations.Meanwhile 42 cases of polycythemia vera patients were collected to detect 2 kinds of JAK2 mutations by the above system and routine methods.Results The multiplex JAK2 mutations detecting system was successfully established by multiplex PCR combined with high-resolution melting curve analysis,which could simultaneously detect JAK2 V617F and JAK2 exon12 mutations.The analytical sensitivities of 2 mutations in this system were both up to 5％ and the precision (coefficient of variation) of intra-and inter-assay of the melting temperature (Tm) of 2 amplicons were separately less than 0.01％.37 cases were identified JAK2 V617F mutations from 42 polycythemia vera patients,while 2 JAK2 exon12 mutations cases were found from 5 JAK2 V617F negative patients.Compared with routine methods,the results matched the rate of 100％.Two cases of JAK2 exon 12 mutations were confirmed to the mutation types of H538K539delinsL and F537-I546dul10 + F547L by cloning and sequencing.Conclusions This method can simultaneously detect two kinds of JAK2 mutations in the peripheral blood and will contribute to the molecular diagnosis of myeloproliferative neoplasms,especially polycythemia vera.