Transgene Detection by Digital Droplet PCR

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（somatic gene therapy）是治疗重症疾病的极具前景的手段。由于其在运动成绩提升方面存在被滥用的潜在可能，世界反兴奋剂机构（World Anti-Doping Agency，WADA）于2004年将‘基因兴奋剂（gene doping）’一词纳入官方禁用物质与方法清单。已有多种基于巢式PCR（nested PCR）或定量PCR（qPCR）的检测策略，旨在检测血液中长期存在的转基因序列，但这些策略均受限于技术瓶颈。本研究开发了一款针对胰岛素样生长因子1（Insulin-Like Growth Factor 1，IGF1）的液滴数字PCR（digital droplet PCR，ddPCR）检测方案，并验证了其应用可行性：对6只经骨骼肌注射携带AAV9-IGF1元件的小鼠及2只对照小鼠开展了为期33天的监测。此外，本研究还构建了可同时检测胰岛素样生长因子1（IGF1）与促红细胞生成素（Erythropoietin，EPO）转基因元件的双重ddPCR检测方案，并建立了全新的DNA提取流程：该流程靶向使用限制性内切酶（restriction enzymes），并包含柱上DNA消化（on-column DNA-digestion）步骤。体内实验数据显示，在为期33天的监测周期内，可从全血提取的DNA中稳定检出IGF1转基因元件。体外实验数据则证实，双重ddPCR可实现高可靠性、高灵敏度的IGF1与EPO检测。柱上DNA消化步骤可显著提升后续基于PCR的检测方法的靶标检出效果。由于ddPCR可实现绝对定量，其具有极佳的日间重复性。因此，我们预期该技术未来可应用于病毒与细菌感染的诊断与监测、突变DNA序列的检测，以及精英运动员体内外源遗传物质存在状况的谱型分析。