Drop-off微滴式数字PCR检测急性髓系白血病NRAS基因突变及其临床应用

Detection of NRAS mutation in acute myeloid leukemia by drop-off droplet digital PCR and its clinical value

目的:建立检测急性髓系白血病(acute myeloid leukemia,AML)神经母细胞瘤RAS病毒癌基因同源物(NRAS)基因突变的drop-off微滴式数字PCR(droplet digital PCR,ddPCR)方法,并评价该方法的临床应用价值。方法:针对NRAS G12和G13突变位点设计特异性drop-off ddPCR引物和探针,建立最佳反应体系,并对该方法进行性能验证。应用该方法对140例临床样本进行初筛,其检测结果与Sanger测序进行比较,并用二代测序(next generation sequencing,NGS)进行验证。结果:建立drop-off ddPCR检测NRAS基因G12/G13突变的方法,灵敏度达0.158%,重复性、线性良好,其空白限为5.40拷贝数/μL,最低检测下限为15.84拷贝数/μL。在140例初诊AML患者中,drop-off ddPCR检出NRAS突变28例(20%),其突变负荷为0.26%~52.85%(中位2.14%);而Sanger测序仅检出7例(5%)阳性,为进一步验证,将21例Sanger测序结果阴性而drop-off ddPCR检测阳性的样本进行NRAS基因NGS,检出14例阳性,而另外7例NGS阴性的样本,其drop-off ddPCR检测突变频率为0.53%~1.50%(中位1.10%)。结论:建立了drop-off ddPCR技术检测AML患者中NRAS基因突变的方法,该方法灵敏度高,可用于对NRAS基因G12/G13突变进行定量检测,有望可用于AML患者预后判断和疾病监测。

Objective:To establish a new drop-off droplet digital PCR(ddPCR)method for detecting NRAS mutations in patients with acute myeloid leukemia(AML),and to evaluate its clinical value.Methods:Specialized primers and probes were designed for NRAS G12 and G13 mutation.This research established and optimized the reaction system of drop-off ddPCR and verified this method.A total of 140 clinical samples were screened by this method,and the results were compared with Sanger sequencing,and verified by next generation sequencing(NGS).Results:Drop-off droplet digital PCR method was established to detect different types of the G12 and G13 mutations of NRAS in AML patients.The method had high sensitivity(up to 0.158%),good repeatability and linearity.The limit of blank was 5.40 copies/μL and the limit of detection was 15.84 copies/μL.Among 140 newly diagnosed AML patients,28 samples(20%)were detected by drop-off ddPCR,the mutation rate was from 0.26%-52.85%(median 2.14%);only 7(5%)samples were detected positive by Sanger sequencing.For further verification,those samples which were detected negatively by Sanger but positively by drop-off ddPCR were sent for NGS.A total of 14 samples were detected positive by NGS,the mutant frequency of the residual 7 detected positive by drop-off ddPCR was 0.53%-1.50%(median 1.10%).Conclusion:Drop-off ddPCR can effectively detect the quantity of the G12 and G13 mutations of NRAS,which can be used for screening and judging the prognosis of AML patients.