采用荧光定量PCR检测JAK2基因V617F突变

Detection of JAK2 V617F mutation by real-time PCR

目的建立荧光定量PCR检测JAK2基因V617F突变的方法，评估JAK2 V617F突变在诊断骨髓增殖性疾病和白血病中的临床意义。方法选取71例慢性粒细胞性白血病（CML）、22例原发性血小板增多症（ET）、11例原发性骨髓纤维化（PMF）、9例真性红细胞增多症（PV）、7例嗜酸粒细胞增多症患者，分别采用荧光定量PCR、突变特异性扩增系统（ARMS）对JAK2 V617F突变进行检测，并采用基因测序对结果进行验证。将具有JAK2 V617F纯合子突变的人类红白血病细胞株（HEL）DNA作为阳性对照，比较荧光定量PCR和ARMS对JAK2基因V617F突变的检测敏感度。结果采用荧光定量PCR检测，野生型的熔解温度（Tm）峰出现于（75．0±0．2）℃处，突变型的Tm峰出现于（76．6±0．2）℃处。JAK2基因V617F突变在PV、ET、PMF等骨髓增殖性疾病中的检出率分别为8例（88．9％）、12例（54．5％）、7例（63．6％），但在71例CML中只检出1例（1．4％）。荧光定量PCR与ARMS的结果符合率为100％，结果经过测序证实。使用荧光定量PCR法可在每10^6个正常白细胞中检出低至10^2个HEL细胞，而使用ARMS方法时要在每10^6个正常白细胞中HEL细胞达到10^4个时，方可检测出，前者比后者方法灵敏100倍。结论成功地建立了荧光定量PCR检测JAK2基因V617F突变的方法，比ARMS更灵敏、简便，适合临床使用。JAK2基因V617F突变在骨髓增殖性疾病中有较高的检出率，可作为骨髓增殖性疾病特异性诊断指标。

Objective To establish real-time PCR method for the detection of JAK2 V617F mutation and evaluate its clinical significance in patients with myeloproliferative disorders and leukemia. Methods 71 chronic myelocytic leukemia （CML） patients, 22 essential thrombocythemia （ET） patients, 11 primary myelofibrosis （PMF） patients, 9 polycythemia vera （PV）patients and 7 eosinophilia patients were enrolled in this study. JAK2 V617F mutation was determined by real-time PCR and amplification refractory mutation system （ ARMS ） , followed by sequencing. Human erythroleukemia cell （ HEL cell） DNA was used as homozygous control of JAK2 V617F mutation. The detection limit for either real-time PCR or ARMS was evaluated. Results Real-time PCR assay showed that there was a melting temperature（Tm） peak at （75.0±0.2） ℃ for wild type samples and a Tm peak at （76.6±0.2） ℃ for mutation type samples. JAK2 V617F mutation was detected in 8（88.9% ） patients with PV, 12（54. 5% ） patients with ET and 7 （63. 6% ） patients with PMF respectively. But there was only one positive case in 71 CML patient（ 1.4% ）. The results showed complete concordance with ARMS results and confirmed by sequencing. The mutation could be detected in 10^2 HEL cells per 10^6 white blood cells by real-time PCR, whereas the mutation can be assessed in 10^4 HEL cells per 10^6 white blood cells by ARMS. Thus, the sensitivity of real-time PCR was 100-fold higher than ARMS. Conclusions The real-time PCR method is successfully established for detection of JAK2 V617F mutation. This method is more sensitive, convenient than ARMS, and suitable for clinical application. There is high frequency of JAK2 V617F mutation in myeloproliferative disorders and it could be used as the diagnostic marker for myeloproliferative disorders.