高灵敏微滴式数字 PCR 检测低水平 JAK2 V617F 突变

目的：

建立一种检测低水平 JAK2 V617F 突变的微滴式数字 PCR (droplet digital PCR,ddPCR) 方法并探讨其在骨髓增殖性肿瘤 (myeloproliferative neoplasm,MPN) 中的应用价值。

方法：

利用位点特异性的 TaqMan-MGB 探针，建立用于检测基因组 DNA 中 JAK2 V617F 突变的 ddPCR 方法，应用该方法、实时定量 PCR 及二代测序方法检测 MPN 患者 JAK2 V617F 突变，比较不同方法检测结果的一致性。

结果：

成功建立了一种基于特异性 TaqMan-MGB 探针检测 JAK2 V617F 突变的 ddPCR 方法，检测灵敏度至少为 0.05 %。应用 ddPCR 方法及实时定量 PCR 方法对 82 份 MPN 患者及 8 份健康对照者的骨髓或外周血 DNA 样本同时进行检测，其中 87 份样本检测结果一致，检出的一致率为 96.7 %；3 份经实时定量 PCR 方法检测为阴性而 ddPCR 检测为阳性的样本，ddPCR 的定量结果分别为 0.015 %、0.002 %、0.039 %, 均低于实时定量 PCR 的检出下限；对于两种方法检测结果均为阳性的 52 份样本，其定量数据的相关系数为 0.9714 (P < 0.0001)。11 例样本同时采用二代测序的方法进行验证，两者定量结果的相关系数为 0.9839 (P < 0.0001)。

结论：

利用特异性 TaqMan-MGB 探针检测 JAK2 V617F 突变的微滴式数字 PCR 方法可便捷、准确地检测 JAK2 V617F 突变，该方法具有高度的灵敏度且可绝对定量，在 JAK2 V617F 突变的筛查及动态监测中具有潜在应用价值。

Objective: 

To establish a droplet digital PCR (ddPCR) method for detecting the low-level JAK2 V617F mutation and to explore its application value in the diagnosis of myeloproliferative neoplasm (MPN).

Methods: 

Site-specific TaqMan-MGB probes were used to establish the ddPCR method for detecting the JAK2 V617F mutation in genomic DNA. JAK2 V617F mutations were detected by ddPCR, real-time quantitative PCR and next-generation sequencing (NGS). The consistency of the detection results of different methods was compared.

Results: 

The ddPCR method for detecting the JAK2 V617F mutation based on specific TaqMan-MGB probes was successfully established, and the detection sensitivity was at least 0.05 %. The ddPCR method and qPCR method were used to detect the JAK2 V617F mutation in 82 subjects with MPN and 8 healthy controls. The consistency rate was 96.7 %. For the 52 samples with positive results by both methods, the correlation coefficient of quantitative data was 0.9714 (P < 0.0001). Eleven samples were verified by NGS and the correlation coefficient between the two quantitative results was 0.9839 (P < 0.0001).

Conclusion: 

The ddPCR with the specific TaqMan-MGB probe method can detect the JAK2 V617F mutation conveniently and accurately, which may have potential application value in the screening and dynamic monitoring of the JAK2 V617F mutation.