用微滴式数字PCR检测母体游离DNA中父源性β-地中海贫血CD41-42突变

Non-invasive prenatal diagnosis for β-thalassemia by detecting paternal CD41-42 mutation in cell-free DNA derived from maternal plasma with droplet digital PCR

目的建立一种用微滴式数字PCR(droplet digital PCR,ddPCR)技术检测母体血浆游离DNA中父源CD41-42突变基因的排除性β-地中海贫血(简称β-地贫)的无创产前诊断方法。方法以β-actin基因为参照序列,β-地贫CD41-42突变基因为目标序列,设计特异性引物和TaqMan探针,建立基于Bio-Rad ddPCR技术的检测体系。通过对目标基因的浓度梯度样本进行检测,评估该方法的准确性、灵敏度和线性检测范围。对20例孕妇的血浆样本进行检测,对该方法进行应用评价。结果建立的ddPCR检测方法能够准确检测出母体血浆游离DNA中的β-地贫CD41-42突变基因的目标序列。目标序列浓度比例为5.00%~0.50%的相对误差均小于0.05,线性回归方程Y=1.0101X-0.0071,R^2=0.9994。20例孕妇血浆游离DNA样本的检测结果与随访确诊结果完全相符。结论建立了一种用ddPCR技术检测母体血浆游离DNA中父源β-地贫CD41-42突变的方法,其操作简单、快速、结果准确、可靠,适用于夫妇为非同型β-地贫基因携带且父方为CD41-42突变的排除性无创产前诊断。

Objective To establish a non-invasive method for β-thalassemia by detecting parental CD41-42 mutation in cell-free DNA derived from maternal plasma with droplet digital PCR (ddPCR). Methods β-actin gene and β-thalassemia gene CD41-42 mutation were respectively set as the reference and target sequences.A novel method was established based on Bio-Rad ddPCR technique with specific primers and TaqMan probes for the two genes.The accuracy,sensitivity and detective Iinearity range of the developed method were evaluated by detection of the target gene gradient concentration samples.The applicability was also evaluated by testing 20maternal plasma samples.Results The ddPCR method could accurately detect the β-thalassemia CD41-42 mutation in cell-free DNA derived from maternal plasma. Within the target sequence concentration ratio of 5.00%-0.50%,the relative errors were all <0.05,the linear regression equation was Y=1.0101X-0.0071 and R^2=0.9994.The results of 20 maternal plasma cell-free DNA samples were all consistent with those of the follow-up testing.Conclusion A ddPCR method for detecting parental CD41-42 mutation in cell-free DNA from maternal plasma was developed.The method is simple,rapid,accurate,and can be applied for non-invasive prenatal diagnosis for couples simultaneously carrying the CD41-42 mutation.