【摘要】 目的：研发一种新的基于TaqMan-MGB探针的实时荧光定量聚合酶链式反应（qPCR）检测方法，用于急性髓系白血病（AML）和慢性中性粒细胞白血病（CNL）患者CSF3R^T618I突变的初筛和随访监测。方法：利用PrimerExpressv3.0软件设计特定的引物和探针，并根据野生型(CSF3R^wt)和突变型(CSF3R^T618I)基因组DNA序列构建质粒标准品，绘制标准曲线，用于定量检测CSF3R^T618I突变。DNA样本来源于421例AML患者和12例CNL患者。结果：成功建立了一种利用TaqMan-MGB探针定量检测CSF3R^T618I突变的方法。在野生型背景下，CSF3R^T618I突变的检测灵敏度可达0.625%。本研究队列中，AML患者的CSF3R^T618I突变检出率为3.33%（14/421）,CNL患者的突变检出率为83.33%（10/12），与高深度靶区测序（TRS）和Sanger测序的结果高度一致。20名健康供者中均未检测到CSF3R^T618I突变。利用标准曲线计算突变阳性的CNL患者CSF3R^T618I等位基因突变频率平均值为（38.90±15.20）%，高于突变阳性AML患者的平均值[（21.72±16.11）%,P=0.015]。结论：基于TaqMan-MGB探针的qPCR是一种简便、灵敏的定量检测方法，可用于筛查CNL或AML患者中的CSF3R^T618I突变，有助于疾病的诊断以及治疗反应的监测。更多还原

【Abstract】 Objective:To develop a new TaqMan-MGB probe based quantitative real-time polymerase chain reaction（qPCR） assay for primary screening and follow-up monitoring of colony-stimulating factor 3 receptor（CSF3R） T618I mutation in patients with acute myeloid leukemia（AML） and chronic neutrophilic leukemia（CNL）.Methods:Specific primers and probes were designed using the Primer Express v3.0 software,and plasmid standards were constructed based on the wild-type(CSF3R^wt) and mutant(CSF3R^T618I) genomic DNA sequences.Standard curve was plotted for the quantitative detection of the hot spot mutation of CSF3R^T618I.DNA samples were obtained from 421 AML and 12 CNL patients.Results:A novel qPCR system based on TaqMan-MGB probes for the quantitative detection of the CSF3R^T618Imutation was successfully established.The predicted sensitivity level was at least 0.625% for CSF3R^T618I mutant allele in a wild-type background.The detection rate of CSF3R^T618I mutation in our cohort was 3.33%（14/421） in AML patients and 83.33%（10/12） in CNL patients,which was highly consistent with the results of high-depth target regional sequencing（TRS） and Sanger sequencing.No CSF3R^T618I mutation was detected in 20 healthy donors.The mean variant allele frequency（VAF） of CSF3R^T618I in mutation-positive CNL patients calculated using the standard curve was（38.90±15.20） %,which was significantly higher than that in mutation-positive AML patients [（21.72±16.11） %,P=0.015].Conclusion:The innovative TaqMan-MGB probe-based qPCR methodology is a reliable,sensitive,and quantitative assay for the detection of CSF3R^T618I mutation in patients with CNL and AML.This approach promises to facilitate the diagnosis of these diseases and enhance the monitoring of therapeutic response.更多还原