【摘要】 目的：建立一种检测低水平JAK2V617F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JAK2V617F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JAK2V617F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JAK2V617F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0.971 4(P<0.000 1)。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0.983 9(P<0.000 1)。结论：利用特异性TaqMan-MGB探针检测JAK2V617F突变的微滴式数字PCR方法可便捷、准确地检测JAK2V617F突变，该方法具有高度的灵敏度且可绝对定量，在JAK2V617F突变的筛查及动态监测中具有潜在应用价值。更多还原

【Abstract】 Objective: To establish a droplet digital PCR(ddPCR) method for detecting the low-level JAK2V617F 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 JAK2V617F mutation in genomic DNA. JAK2V617F 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 JAK2V617F 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 JAK2V617F 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 0.971 4(P<0.000 1). Eleven samples were verified by NGS and the correlation coefficient between the two quantitative results was 0.983 9(P<0.000 1). Conclusions: The ddPCR with the specific TaqMan-MGB probe method can detect the JAK2V617F mutation conveniently and accurately, which may have potential application value in the screening and dynamic monitoring of the JAK2V617F mutation.更多还原