Objective: To develop a new method based on droplet digital PCR (DD-PCR) for detection and quantification of maternal cell contamination in prenatal diagnosis. Methods: Invasive prenatal samples from 40 couples of β(IVS-Ⅱ-654)/β(N) thalassemia gene carriers who accepted prenatal diagnosis in Affiliated Women and Children's Hospital of Guangzhou Medical University from October 2015 to December 2016 were analyzed retrospectively. Specific primers and probes were designed. The concentration gradient were 50%, 25%, 12.5%, 6.25%, 3.125%, 1.562 5%. There were 40 groups of prenatal diagnostic samples. Comparing DD-PCR with quantitative fluorescent-PCR (QF-PCR) based on the short tandem repeats for assement of the sensitivity and accuracy of maternal cell contamination, respectively. Results: DD-PCR could quantify the maternal cell contamination as low as 1.562 5%. The result was proportional to the dilution titers. In the 40 prenatal samples, 6 cases (15%, 6/40) of maternal cell contamination were detected by DD-PCR, while the QF-PCR based on short tandem repeat showed 3 cases (7.5%, 3/40) with maternal cell contamination, DD-PCR was more accurate (P=0.002) . Conclusion: DD-PCR is a precise and sensitive method in the detection of maternal cell contamintation. It could be useful in clinical application.
目的： 探讨微滴式数字PCR（DD-PCR）技术检测产前诊断标本母体细胞污染的临床应用价值。 方法： 2015年10月至2016年12月间以40例因夫妇双方均为地中海贫血基因β(IVS-Ⅱ-654)/β(N)杂合携带者、在广州医科大学附属广东省妇女儿童医院行侵入性产前诊断的孕妇作为观察样本。通过设计特异性引物、探针，建立母体细胞污染程度梯度50%、25%、12.5%、6.25%、3.125%、1.562 5%模型，采用DD-PCR技术及短串联重复序列的荧光定量PCR技术检测产前诊断标本母体细胞污染，分别评估两种检测方法的敏感性；并分别检测40组产前诊断标本，以比较DD-PCR技术与荧光定量PCR技术检测母体细胞污染的准确性。 结果： DD-PCR技术可以检测到产前诊断标本中低至1.562 5%的母体细胞污染，检测结果与母体细胞污染程度梯度改变基本相符，而荧光定量PCR技术只能在母体细胞污染≥6.25%时才提示产前诊断标本存在400 bp的母源性特有峰。DD-PCR技术共检测到6例（15%，6/40）产前诊断标本存在母体细胞污染，荧光定量PCR技术只检测到3例母体细胞污染的产前诊断标本（7.5%，3/40），两种检测方法比较，DD-PCR技术的准确性更高（P=0.002）。 结论： DD-PCR技术可对产前诊断标本进行母体细胞污染精确定量，并具有较高的敏感性，具有良好的临床应用价值。.
Keywords: DNA contamination; Genotype; Polymerase chain reaction; Prenatal diagnosis; Thalassemia.