Prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes using amniotic fluid as starting material of PCR
Prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes using amniotic fluid as starting material of PCR
复制标题
使用羊水作为 PCR 起始材料，通过对杂合子进行定量焦磷酸测序来进行 21 三体性产前诊断。
DOI：
10.1039/c3an36903j
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发表时间：
2013-01-01
期刊：
影响因子：
4.2
通讯作者：
Zhou，Guohua
中科院分区：
文献类型：
--
作者：
Ye，Hui;Wu，Haiping;Zhou，Guohua
通过MALDI-TOF质谱（MS），SNP的等位基因比率已用于胎儿21三体的产前诊断。由于MALDI-TOF MS在定量分析性能方面具有挑战性，因此提出焦磷酸测序来代替MS以更好地定量分析等位基因比率。为了实现简单和快速的临床诊断，开发了使用高pH缓冲液（HpH缓冲液）的PCR，以直接扩增羊水，而无需任何基因组DNA提取步骤。通过建立的检测方法，114份羊水样本直接扩增，并通过焦磷酸测序每个样本的5个SNP进行单独分析;从而计算整倍体杂合子的等位基因比率，以确定产前诊断21三体的临界值。五个SNP的组在杂合性方面高得多，并且在114个样品中的每一个中发现至少一个杂合子，并且86%的样品在组中具有至少两个杂合子，给出了接近100%的测定灵敏度。应用该阈值对20例临床样本进行21三体检测，置信度均在99%以上，表明该方法与核型分析结果一致。总之，这种基于焦磷酸测序的方法，加上羊水的直接扩增，是准确的定量基因分型，操作简单。我们相信，这种方法可能是一个有前途的替代染色体核型分析在产前诊断。
Allelic ratio of an SNP has been used for prenatal diagnosis of fetal trisomy 21 by MALDI-TOF mass spectrometry (MS). Because MALDI-TOF MS is challenging in quanepsication performance, pyrosequencing was proposed to replace MS for better quanepsication of allelic ratios. To achieve a simple and a rapid clinical diagnosis, PCR with a high-pH buffer (HpH buffer) was developed to directly amplify amniotic fluid without any step of genomic DNA extraction. By the established assay, 114 samples of amniotic fluid were directly amplified and individually analyzed by the pyrosequencing of five SNPs of each sample; the allelic ratios of euploid heterozygotes were thus calculated to determine the cutoff values of prenatal diagnosis of trisomy 21. The panel of five SNPs is much high in heterozygosity, and at least one heterozygote was found in each of the 114 samples, and 86 % of the samples have at least two heterozygotes in the panel, giving a nearly 100 % sensitivity of the assay. By using the cutoff values of each SNP, 20 pre-diagnosed clinical samples were detected as trisomy 21 carriers with the confidence level over 99 %, indicating that our method and karyotyping analysis are consistent in results. In conclusion, this pyrosequencing-based approach, coupled with the direct amplification of amniotic fluid, is accurate in quantitative genotyping and is simple in operation. We believe that the approach could be a promising alternative to karyotyping analysis in prenatal diagnosis.