Department of Sports Medicine, Medical Clinic, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
Gene Ther. 2011 Mar;18(3):225-31. doi: 10.1038/gt.2010.122. Epub 2010 Sep 2.
The misuse of somatic gene therapy for the purpose of enhancing athletic performance is perceived as a coming threat to the world of sports and categorized as 'gene doping'. This article describes a direct detection approach for gene doping that gives a clear yes-or-no answer based on the presence or absence of transgenic DNA in peripheral blood samples. By exploiting a priming strategy to specifically amplify intronless DNA sequences, we developed PCR protocols allowing the detection of very small amounts of transgenic DNA in genomic DNA samples to screen for six prime candidate genes. Our detection strategy was verified in a mouse model, giving positive signals from minute amounts (20 μl) of blood samples for up to 56 days following intramuscular adeno-associated virus-mediated gene transfer, one of the most likely candidate vector systems to be misused for gene doping. To make our detection strategy amenable for routine testing, we implemented a robust sample preparation and processing protocol that allows cost-efficient analysis of small human blood volumes (200 μl) with high specificity and reproducibility. The practicability and reliability of our detection strategy was validated by a screening approach including 327 blood samples taken from professional and recreational athletes under field conditions.
出于提高运动成绩的目的而错误使用体细胞核移植技术被视为对体育界的一种潜在威胁，并被归类为“基因兴奋剂”。本文描述了一种直接检测基因兴奋剂的方法，该方法基于外周血样中转基因 DNA 的存在与否，给出明确的是/否答案。通过利用一种引物策略来特异性扩增无内含子的 DNA 序列，我们开发了 PCR 方案，允许在基因组 DNA 样本中检测极少量的转基因 DNA，以筛选六个主要候选基因。我们的检测策略在小鼠模型中得到了验证，在肌肉内腺相关病毒介导的基因转移后长达 56 天的时间里，从微量（20μl）血液样本中得到了阳性信号，这是最有可能被滥用于基因兴奋剂的候选载体系统之一。为了使我们的检测策略适用于常规检测，我们实施了一种稳健的样品制备和处理方案，允许以高特异性和可重复性分析小体积的人血（200μl）。通过在现场条件下对 327 名专业和业余运动员的血液样本进行筛选，验证了我们的检测策略的实用性和可靠性。
