Optimization of Transrenal DNA Analysis: Detection of Fetal DNA in Maternal Urine
Optimization of Transrenal DNA Analysis: Detection of Fetal DNA in Maternal Urine
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DOI：
10.1373/clinchem.2008.113050
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发表时间：
2009-04-01
影响因子：
9.3
通讯作者：
Umansky, Samuil R.
中科院分区：
文献类型：
--
作者：
Shekhtman, Eugene M.;Anne, Kalpana;Umansky, Samuil R.
背景：来自全身死亡细胞的DNA片段可在尿液中检测到（transrenal DNA，或Tr-DNA）。我们的目标是Tr-DNA的分离和检测技术的优化，作为一个模型的孕妇urina.METHODS胎儿DNA的分析：我们分离尿DNA使用传统的二氧化硅为基础的方法，并使用一种新的技术的基础上吸附的无细胞核酸的Q-Sepharose树脂。采用常规PCR和实时荧光定量PCR方法检测孕妇尿中Y染色体特异性SRY（sex-determining region Y，性别决定区Y）序列，并对25、39、65和88 bp PCR靶序列设计引物/探针。检测诊断灵敏度随着PCR靶缩短而增加。用Q树脂法可以分离出较短的DNA片段（50-150 bp），这也有利于胎儿Tr-DNA的分析。利用Q树脂法提取的DNA和“超短”DNA靶，我们成功地检测了82例怀有男性胎儿的孕妇的78例尿液样本中的SRY序列（阳性预测值87.6%）。91例孕妇尿标本中有11例SRY假阳性（阴性预测值95.2%）。结论：采用适当的DNA分离和分析方法，可以成功地从孕妇尿中检测出单拷贝胎儿DNA序列。为了避免假阳性结果，有必要采取强有力的预防措施，防止样本被雄性细胞和DNA污染。(C)2008年美国临床化学协会
BACKGROUND: Fragments of DNA from cells dying throughout the body are detectable in urine (transrenal DNA, or Tr-DNA). Our goal was the optimization of Tr-DNA isolation and detection techniques, using as a model the analysis of fetal DNA in maternal urine.METHODS: We isolated urinary DNA using a traditional silica-based method and using a new technique based on adsorption of cell-free nucleic acids on Q-Sepharose resin. The presence of Y chromosome-specific SRY (sex-determining region Y) sequences in urine of pregnant women was detected by conventional and real-time PCR using primers/probe sets designed for 25-, 39-, 65-, and 88-bp PCR targets.RESULTS: Method of DNA isolation and PCR target size affected fetal Tr-DNA detection. Assay diagnostic sensitivity increases as the PCR target is shortened. Shorter DNA fragments (50-150 bp) could be isolated by Q-resin-based technique, which also facilitated fetal Tr-DNA analysis. Using DNA isolated by Q-resin-based method and an "ultrashort" DNA target, we successfully detected SRY sequences in 78 of 82 urine samples from women pregnant with male fetuses (positive predictive value 87.6%). Eleven of 91 urine samples from women pregnant with female fetuses produced SRY false-positive results (negative predictive value 95.2%).CONCLUSIONS: Single-copy fetal DNA sequences can be successfully detected in the urine of pregnant women when adequate methods for DNA isolation and analysis are applied. Strong precautions against sample contamination with male cells and DNA are necessary to avoid false-positive results. (C) 2008 American Association for Clinical Chemistry