Rice John E, Sanchez J Aquiles, Pierce Kenneth E, Wangh Lawrence J
Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA.
Prenat Diagn. 2002 Dec;22(12):1130-4. doi: 10.1002/pd.500.
The results presented here provide the first single-cell genetic assay for Tay-Sachs disease based on real-time PCR. Individual lymphoblasts were lysed with an optimized lysis buffer and assayed using one pair of primers that amplifies both the wild type and 1278 + TATC Tay-Sachs alleles. The resulting amplicons were detected in real time with two molecular beacons each with a different colored fluorochrome. The kinetics of amplicon accumulation generate objective criteria by which to evaluate the validity of each reaction. The assay had an overall utility of 95%, based on the detection of at least one signal in 235 of the 248 attempted tests and an efficiency of 97%, as 7 of the 235 samples were excluded from further analysis for objective quantitative reasons. The accuracy of the assay was 99.1%, because 228 of 230 samples gave signals consistent with the genotype of the cells. Only two of the 135 heterozygous samples were allele drop-outs, a rate far lower than previously reported for single-cell Tay-Sachs assays using conventional methods of PCR.
本文展示的结果提供了首个基于实时聚合酶链反应（PCR）的泰-萨克斯病单细胞基因检测方法。使用优化的裂解缓冲液裂解单个淋巴母细胞，并使用一对引物进行检测，该引物可扩增野生型和1278 + TATC泰-萨克斯等位基因。用两个带有不同颜色荧光染料的分子信标实时检测产生的扩增子。扩增子积累的动力学产生了评估每个反应有效性的客观标准。基于在248次尝试检测中的235次检测到至少一个信号，该检测方法的总体效用为95%，效率为97%，因为235个样本中有7个因客观定量原因被排除在进一步分析之外。该检测方法的准确性为99.1%，因为230个样本中有228个给出的信号与细胞基因型一致。135个杂合样本中只有两个出现等位基因缺失，这一比例远低于此前使用传统PCR方法进行单细胞泰-萨克斯检测所报告的比例。
