Department of Obstetrics and Gynecology, Research Institute of Surgery, Daping Hospital, Third Military Medical University, 10 Changjiangzhilu, Daping, Yuzhong District, Chongqing 400042, People's Republic of China.
Cell Biochem Biophys. 2011 Dec;61(3):629-36. doi: 10.1007/s12013-011-9248-7.
The aim of this study was to investigate the feasibility of combining PCR and ligase detection reaction (LDR) with a novel nano-gold-based universal array for the detection of low abundance point mutations from fetal DNA in maternal plasma samples. The sequence with the target point mutation was first amplified by PCR and then used as a template for LDR in which the upstream specific primer contains a tag sequence at the 5'-end. After hybridization to the probes of a universal array containing anti-tag sequences, the ligated products were bound to streptavidin-labeled nano-gold particles and the hybridization signals were amplified by silver staining. The PCR/LDR/universal array was first tested for sensitivity with nano-gold-based detection, and then this system was applied to detect the low abundance specific mutation IVS2 654(C→T) of the β-globin gene in a model using maternal plasma samples. The nano-gold-based method unambiguously identified a single mutation at a sensitivity of 1:1000. This approach was applied to detect the paternally inherited IVS2 654(C→T) mutation from thirty maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. The PCR/LDR/nano-gold-based universal array is able to detect low-abundance point mutations with high sensitivity.
本研究旨在探索一种将 PCR 和连接酶检测反应 (LDR) 与新型纳米金通用阵列相结合，从母体血浆样本中检测低丰度点突变的可行性。首先通过 PCR 扩增具有靶点点突变的序列，然后将其作为 LDR 的模板，其中上游特异性引物在 5'端包含一个标记序列。与包含抗标记序列的通用阵列的探针杂交后，连接产物与链霉亲和素标记的纳米金颗粒结合，并通过银染放大杂交信号。首先使用纳米金检测对 PCR/LDR/通用阵列进行了敏感性测试，然后将该系统应用于使用母体血浆样本检测 β-珠蛋白基因的低丰度特异性突变 IVS2 654(C→T)的模型。纳米金方法能够以 1:1000 的灵敏度明确识别单个突变。该方法应用于从 30 个母体血浆样本中检测父系遗传的 IVS2 654(C→T)突变。结果与羊水细胞 DNA 的 PCR/反向斑点印迹的结果一致。PCR/LDR/纳米金通用阵列能够以高灵敏度检测低丰度点突变。
