Moser Dirk A, Braga Luca, Raso Andrea, Zacchigna Serena, Giacca Mauro, Simon Perikles
Faculty of Psychology, Genetic Psychology, Ruhr-University-Bochum, Bochum, Germany; Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg-University Mainz, Mainz, Germany.
International Centre for Genetic Engineering and Biotechnology (ICGEB), Molecular Medicine, Trieste, Italy.
PLoS One. 2014 Nov 6;9(11):e111781. doi: 10.1371/journal.pone.0111781. eCollection 2014.
Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.
体细胞基因治疗是治疗严重疾病的一种有前景的工具。由于其在体育领域存在被滥用以提高成绩的可能性，世界反兴奋剂机构（WADA）于2004年将“基因兴奋剂”一词列入违禁物质和方法的官方清单。已经提出了几种基于巢式PCR或qPCR的策略，旨在检测转基因在血液中的长期存在，但这些策略受到技术限制的阻碍。我们开发了一种用于检测胰岛素样生长因子1（IGF1）的数字液滴PCR（ddPCR）方案，并证明了其在33天内监测6只注射了AAV9-IGF1元件的骨骼肌小鼠和2只对照小鼠的适用性。创建了一种用于同时检测胰岛素样生长因子1（IGF1）和促红细胞生成素（EPO）转基因元件的双重ddPCR方案。建立了一种新的DNA提取程序，包括柱上DNA消化在内，以靶向方式使用限制性酶。体内数据显示，在从全血中提取的DNA中，IGF1转基因元件在33天内可以可靠地检测到。体外数据表明，通过双重ddPCR检测IGF1和EPO具有很高的可靠性和灵敏度。柱上DNA消化在基于PCR的下游方法中显著提高了目标检测。由于ddPCR提供绝对定量，它确保了出色的日常重现性。因此，我们预计这项技术未来将用于诊断和监测病毒和细菌感染、检测突变的DNA序列以及检测精英运动员中外源遗传物质的存在情况。
