Yi Ping, Chen Zhuqin, Yu Lili, Zheng Yingru, Liu Guodong, Xie Haichang, Zhou Yuanguo, Zheng Xiuhui, Han Jian, Li Li
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.
J Matern Fetal Neonatal Med. 2010 Aug;23(8):920-7. doi: 10.3109/14767050903387060.
Analysis of fetal DNA in maternal plasma has recently been introduced for non-invasive prenatal diagnosis. We have now investigated the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of fetal point mutations, such as the beta-thalassemia mutation, IVS2 654(C --> T), in maternal plasma DNA.
The sensitivity of LDR/capillary electrophoresis was examined by quantifying the mutant PCR products in the presence of a vast excess of non-mutant competitor template, a situation that mimics the detection of rare fetal mutations in the presence of excess maternal DNA. PCR/LDR/capillary electrophoresis was applied to detect the mutation, IVS2 654(C --> T), in an experimental model at different sensitivity levels and from 10 maternal plasma samples.
Our results demonstrated that this approach to detect a low abundance IVS2 654(C --> T) mutation achieved a sensitivity of approximately 1:10,000. The approach was applied to maternal plasma DNA to detect the paternally inherited fetal IVS2 654(C --> T) mutation, and the results were equivalent to those obtained by PCR/reverse dot blot of amniotic fluid cell DNA.
PCR/LDR/capillary electrophoresis has a very high sensitivity that can distinguish low abundance single nucleotide differences and can detect paternally inherited fetal point mutations in maternal plasma.
母体血浆中胎儿DNA分析最近已被用于非侵入性产前诊断。我们现在研究了聚合酶链反应（PCR）/连接酶检测反应（LDR）/毛细管电泳用于检测母体血浆DNA中胎儿点突变（如β地中海贫血突变IVS2 654（C→T））的可行性。
通过在大量非突变竞争模板存在的情况下对突变PCR产物进行定量来检测LDR/毛细管电泳的灵敏度，这种情况模拟了在过量母体DNA存在下检测罕见胎儿突变的情况。将PCR/LDR/毛细管电泳应用于不同灵敏度水平的实验模型以及10份母体血浆样本中，以检测IVS2 654（C→T）突变。
我们的结果表明，这种检测低丰度IVS2 654（C→T）突变的方法灵敏度约为1:10,000。该方法应用于母体血浆DNA以检测父系遗传的胎儿IVS2 654（C→T）突变，结果与通过羊水细胞DNA的PCR/反向斑点杂交获得的结果相当。
PCR/LDR/毛细管电泳具有非常高的灵敏度，能够区分低丰度单核苷酸差异，并且可以检测母体血浆中父系遗传的胎儿点突变。
