《中国生物工程杂志》
2023年第12期
龙玲玉,周亚兰,焦瑶,叶林海,李修春,刘艳荣,黄晓军,阮国瑞
Abstract
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.
Tags:
骨髓增殖性肿瘤(Myeloproliferative neoplasms);JAK2 V617F突变(JAK2 V617F mutation); 微滴式数字PCR(Droplet digital PCR)