Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2 V617F Mutation

IF 1.2 Q4 GENETICS & HEREDITY
Yupeng Liu, Cong Han, Jie Li, Shicai Xu, Z. Xiao, Zhiyun Guo, Shuquan Rao, Yao Yao
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引用次数: 0

Abstract

Precise quantification of the JAK2 V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2 V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2 V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations.
实验室开发的用于定量检测 JAK2 V617F 突变的液滴数字 PCR 检测试剂盒
使用高灵敏度的检测方法精确量化 JAK2 V617F 突变对于骨髓增生性肿瘤 (MPN) 患者的诊断、治疗过程监控和预后预测至关重要。数字液滴聚合酶链反应(ddPCR)能在高比例的野生型等位基因中精确定量低水平突变,而无需外部校准物或内源性对照。本研究的目的是优化检测 JAK2 V617F 突变的 ddPCR 检测方法,并将其确立为本中心实验室开发的 ddPCR 检测方法。优化过程包括微调五个关键参数:引物/探针序列和浓度、退火温度、模板量和 PCR 循环。我们的 ddPCR 检测方法具有极高的灵敏度,定量限(LoQ)为 0.01% 的变异等位基因频率,变异系数约为 76%。对 39 份样本进行的定量 PCR 比较分析表明其一致性极佳(r = 0.988)。总之,通过严格的优化过程和全面的分析性能验证,我们建立了实验室开发的用于检测 JAK2 V617F 的高灵敏度和高分辨力的 ddPCR 平台。这种优化的检测方法有望在骨髓增生性疾病患者和非骨髓增生性疾病人群中用于早期检测极小残留病、个性化风险分层以及更有效的治疗策略。
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来源期刊
Global Medical Genetics
Global Medical Genetics GENETICS & HEREDITY-
自引率
11.80%
发文量
30
审稿时长
14 weeks
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