Akonni Biosystems, Inc, Frederick, Maryland, United States of America.
PLoS One. 2013 Aug 6;8(8):e73068. doi: 10.1371/journal.pone.0073068. Print 2013.
Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed--a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods.
由于在妊娠早期母体血浆中存在的胎儿 DNA 比例较低（<10%），因此需要有效的提取方法来成功应用于非侵入性产前诊断检测的下游检测。在这项研究中，比较了两种使用相似化学原理但不同工作流程的提取方法，以比较其分离效率和胎儿 DNA 回收率。Akonni Biosystems 的 TruTip 技术使用嵌入在吸头中的结合基质；Qiagen 的 Circulating Nucleic Acids Kit 采用离心柱方法。TruTip 方法增加了一个额外的步骤，从样品中减少大于 600bp 的 DNA 片段的回收，从而产生更高比例的较小分子量 DNA，有效地富集胎儿 DNA。在这项评估中，进行了三项独立的提取比较研究——血浆中碎片化 DNA 的稀释系列、一组临床母体样本以及母体样本的血液采集管时间点研究。两种提取方法都能从大量血浆中有效地提取小片段 DNA。在修改后的样品中，TruTip 提取方法的总体 DNA 回收率低约 15%，但与 Qiagen 方法相比，胎儿 DNA 的百分比增加了 87%。与 Qiagen 方法相比，TruTip 提取的样本中胎儿 DNA 的平均百分比增加了 55%，适用于所有组的盲法临床样本。比较在处理成血浆之前孵育长达 48 小时的全血样本的提取效率的研究表明，TruTip 方法的胎儿 DNA 回收率更一致。提取产物在两种检测平台上进行了测试，定量实时 PCR 和液滴数字 PCR，两种提取方法均产生了相似的结果。
