Sensitive and accurate quantification of JAK2 V617F mutation in chronic myeloproliferative neoplasms by droplet digital PCR
Sensitive and accurate quantification of JAK2 V617F mutation in chronic myeloproliferative neoplasms by droplet digital PCR
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DOI：
10.1007/s00277-016-2623-0
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发表时间：
2016-04-01
影响因子：
3.5
通讯作者：
Finke, Juergen
中科院分区：
文献类型：
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作者：
Waterhouse, Miguel;Follo, Marie;Finke, Juergen
JAK 2 V617 F突变在骨髓增生性肿瘤（MPN）患者中的检出率很高。MPN治疗效率可以通过JAK 2 V617 F定量来评估。实时定量PCR（qPCR）广泛用于JAK 2 V617 F定量。已经描述了新兴的替代技术，如数字液滴PCR（ddPCR），以克服固有的qPCR限制。本研究的目的是评价ddPCR在MPN患者样本中用于JAK 2 V617 F定量的效用。用人工DNA混合物建立敏感性和特异性。此外，对来自59名患者的101份样本进行了JAK 2 V617 F突变评估。qPCR和ddPCR的检测限均为0.01%。通过两种PCR平台在59名患者中的43名中检测到JAK 2 V617 F突变。然而，在14%的样本中，JAK 2 V617 F突变仅用ddPCR检测到。这14%的不一致样本来自异基因干细胞移植后不久的患者。临床样品中通过qPCR和ddPCR测量的JAK 2 V617 F突变的百分比显示出高度相关性（斯皮尔曼r：0.9637 p < 0.001）和通过Bland-Altman分析评估的极好的一致性。总之，ddPCR是定量JAK 2 V617 F突变的合适、精密和灵敏的方法。
The JAK2 V617F mutation can be detected with a high frequency in patients with myeloproliferative neoplasms (MPN). MPN treatment efficiency can be assessed by JAK2 V617F quantification. Real-time quantitative PCR (qPCR) is widely used for JAK2 V617F quantification. Emerging alternative technologies like digital droplet PCR (ddPCR) have been described to overcome inherent qPCR limitations. The purpose of this study was to evaluate the utility of ddPCR for JAK2 V617F quantification in patient samples with MPN. Sensitivity and specificity were established by using DNA artificial mixtures. In addition, 101 samples from 59 patients were evaluated for JAK2 V617F mutation. Limit of detection was 0.01 % for both qPCR and ddPCR. The JAK2 V617F mutation was detected in 43 out of 59 patients by both PCR platforms. However, in 14 % of the samples, JAK2 V617F mutation was detected only with ddPCR. This 14 % of discrepant samples were from patients shortly after allogeneic stem cell transplantation. Percentage of JAK2 V617F mutation measured by qPCR and ddPCR in clinical samples showed a high degree of correlation (Spearman r: 0.9637 p < 0.001) and an excellent agreement assessed by Bland-Altman analysis. In conclusion, ddPCR is a suitable, precise, and sensitive method for quantification of the JAK 2 V617F mutation.