Rajan-Babu Indhu-Shree, Lian Mulias, Tran Anh H, Dang Truong T, Le Huong T-M, Thanh Minh N, Lee Caroline G, Chong Samuel S
Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore.
J Mol Diagn. 2016 Sep;18(5):719-730. doi: 10.1016/j.jmoldx.2016.05.002. Epub 2016 Jul 1.
Population-based screening for CGG-repeat expansions in the fragile X mental retardation 1 (FMR1) gene that cause fragile X syndrome can now be performed more cost-effectively and simply by combining direct triplet-primed PCR (dTP-PCR) with melting curve analysis (MCA). We have now performed a detailed technical validation to define the operational parameters for achieving robust and reliable performance of the FMR1 dTP-PCR MCA assay. We compared the assay's performance on 2 real-time PCR platforms and determined its analytic sensitivity and specificity. We also assessed the assay's performance on DNA isolated from different sources, the effect of differences in CGG-repeat length and AGG-interruption pattern on melt peak temperature (Tm), and the effect of common substances found in DNA solutions on Tms. The assay performed well in distinguishing normal from expansion-carrying samples. The assay had detection sensitivity down to 1 ng and an analytical specificity beyond 150 ng. In addition to peripheral blood DNA, analysis could also be performed on DNA from saliva, buccal swabs, and dried blood spots. Salt increased Tms, glycogen contamination had minimal effect, whereas AGG interruptions lowered Tms. The FMR1 dTP-PCR MCA screening assay is highly sensitive and specific, performs well using DNA from different sources, and is robust and reproducible when reagent concentrations are maintained across all tested samples.
通过将直接三联体引物PCR（dTP-PCR）与熔解曲线分析（MCA）相结合，现在可以更具成本效益且更简便地对导致脆性X综合征的脆性X智力低下1（FMR1）基因中的CGG重复扩增进行基于人群的筛查。我们现在已经进行了详细的技术验证，以确定实现FMR1 dTP-PCR MCA检测稳健可靠性能的操作参数。我们比较了该检测在2个实时PCR平台上的性能，并确定了其分析灵敏度和特异性。我们还评估了该检测在从不同来源分离的DNA上的性能、CGG重复长度和AGG中断模式差异对熔解峰温度（Tm）的影响，以及DNA溶液中常见物质对Tm的影响。该检测在区分正常样本和携带扩增的样本方面表现良好。该检测的检测灵敏度低至1 ng，分析特异性超过150 ng。除了外周血DNA外，还可以对唾液、口腔拭子和干血斑中的DNA进行分析。盐会提高Tm，糖原污染影响最小，而AGG中断会降低Tm。FMR1 dTP-PCR MCA筛查检测具有高度的敏感性和特异性，使用来自不同来源的DNA表现良好，并且当所有测试样本的试剂浓度保持一致时，该检测稳健且可重复。
