采用微阵列数字PCR检测JAK2基因V617F突变  

Detecting JAK2 V617F mutation in myeloproliferative neoplasms by microarray based digital PCR

作　　者：许笑[1] 张群峰 张心菊[1] 唐宜桂[3] 任惠民[1] 杨瑞[4] 樊妮[5] 陈波斌[5] 关明[1] 

机构地区：[1]复旦大学附属华山医院中心实验室,上海200040 [2]上海市第五人民医院检验科 [3]复旦大学附属华山医院检验科 [4]复旦大学附属金山医院 [5]复旦大学附属华山医院血液科

出　　处：《中华检验医学杂志》2016年第3期176-180,共5页Chinese Journal of Laboratory Medicine

基　　金：上海申康医院发展中心市级医院临床辅助科室能力建设项目（SHDC22014001）;2015年度上海科委“创新行动计划”基础研究项目（15JC1401100）

摘　　要：目的采用数字PCR检测骨髓增殖性肿瘤相关的JAK2基因V617F突变，评估该法的灵敏度、重复性和准确性。方法方法学评价。白2014—2015年间复旦大学附属华山医院收治的患者中，选取已知JAK2基因V617F突变的18例真性红细胞增多症病例、11例原发性血小板增多症病例、2例特发性骨髓纤维化病例及未知突变的6例红细胞异常增高病例和10名健康对照者。分别以HEL细胞株DNA和SW480细胞株DNA为突变型对照和野生型对照，稀释突变标准品为30％、10％、1％、0．1％和0．01％以验证微阵列数字PCR体系的灵敏度。使用两例低突变负荷样本（1％和10％），各重复5次，验证数字PCR体系重复性。以MGB探针法荧光定量PCR作为对照参考方法，检测微阵列数字PCR的检测性能。结果实验结果表明两种检测方法相关性高（R^2=0．9983），而微阵列数字PCR能够检测最低约0．1％的微量突变，相应的突变拷贝数绝对定量为0．16拷贝／μl。突变负荷为1％和10％的样本重复性检测结果CV分别为17．29％和7．50％。此外，MGB探针法和数字PCR法均成功地从31例已知突变的病例样本中均检出JAK2基因V617F突变阳性，而10名健康对照者都为阴性，两种方法得到的结果是一致的。从6例红细胞异常增高的病例中。MGB法未检出突变，而数字PCR成功检出2例微量突变样本，其突变率分别为0．37％和0．18％。结论微阵列数字PCR在JAK2基因V617F突变检测体系中表现出比荧光定量PCR的更高的灵敏度以及良好的稳定性，有助于其在体细胞突变微量情况下更准确地检出突变，避免漏诊误诊。Objective To evaluate the sensitivity, repeatability and accuracy of mieroarray digital PCR system in detecting JAK2 V617F mutation, which was closely related to myeloproliferative neoplasms （MPN）. Methods All of the 31 MPN patients with JAK2 V617F mutation, including 18 cases of polycythemia vera （PVs） , 11 primary thrombocythemias （ETs） and 2 primary inyelofibrosis （ PMFs ） , were collected fi＇om Huashan Hospital, Fudan University during 2014 - 2015, while 10 normal controls and 6 cases with abnormal increased hemoglobin were involved. Human erylhroleukemia cell line （HEL） and eoloreetal cancer cell SW480 were used as the mutant and the wild type control, respectively. The sensitivity of microan＇ay digital PCR were verified by detecting the gradient diluted mutation standard harboring 30% , 10% , 1% , 0.1% and 0.01% mutant allele burden, respectively . Repeatability was evaluated by detecting 1% and 10% mutated samples for 5 times, respectively. MGB probe real time PCR was selected as the reference method to verify the accuracy of the digital PCR. Results With digital PCR, the accurate quantitation of JAK2 V617F mutation was achieved down to 0.1% , which is approximate to 0.16 copies per microliter. The results obtained from the two kinds of technique showed a high correlation by linear regression analysis （R2 = O. 998 3 ）. The results of repeated samples showed CVs as 17. 18% for 1% mutant allele burden and 7.50% for 10%. Among all cases, the 31 patients known mutated were detected as positive and 10 controls as negative by both digital PCR and Real time PCR. In another 6 cases, 2 were found JAK2 V617F mutation of low allele burdens of 0.37% and 0.18% by digital PCR but detected as negative by real time PCR. Conclusions Microarray digital PCR offers a higher sensitivity and better repeatability than real time PCR which could help detect rare JAK2 V617F mutations in MPNs accurately.

关 键 词：聚合酶链反应 微芯片分析技术 DNA突变分析 骨髓肿瘤 

分 类 号：R55[医药卫生—血液循环系统疾病] R440[医药卫生—内科学]