Gene doping confers health risks for athletes and is a threat to fair competition in sports. Therefore the anti-doping community has given attention on its detection. Previously published polymerase chain reaction-based methodologies for gene doping detection are targeting exon-exon junctions in the intron-less transgene. However, because these junctions are known, it would be relatively easy to evade detection by tampering with the copyDNA sequences. We have developed a targeted next-generation sequencing based assay for the detection of all exon-exon junctions of the potential doping genes, EPO, IGF1, IGF2, GH1, and GH2, which is resistant to tampering. Using this assay, all exon-exon junctions of copyDNA of doping genes could be detected with a sensitivity of 1296 copyDNA copies in 1000 ng of genomic DNA. In addition, promotor regions and plasmid-derived sequences are readily detectable in our sequence data. While we show the reliability of our method for a selection of genes, expanding the panel to detect other genes would be straightforward. As we were able to detect plasmid-derived sequences, we expect that genes with manipulated junctions, promotor regions, and plasmid or virus-derived sequences will also be readily detected.

中文翻译：

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一种用于基因掺杂检测的新一代测序方法，可将低水平的质粒 DNA 与基因组 DNA 的背景区分开来。

基因兴奋剂会给运动员带来健康风险，并威胁到体育运动的公平竞争。因此，反兴奋剂界对其检测给予了重视。以前发表的基于聚合酶链反应的基因掺杂检测方法是针对无内含子转基因中的外显子-外显子连接。然而，由于这些连接点是已知的，通过篡改拷贝DNA序列来逃避检测将相对容易。我们开发了一种基于下一代测序的靶向检测，用于检测潜在的兴奋剂基因 EPO、IGF1、IGF2、GH1 和 GH2 的所有外显子-外显子连接，它具有抗篡改能力。使用该测定法，可以检测到掺杂基因的 copyDNA 的所有外显子-外显子连接，灵敏度为 1000 ng 基因组 DNA 中的 1296 个 copyDNA 拷贝。此外，在我们的序列数据中很容易检测到启动子区域和质粒衍生序列。虽然我们展示了我们的方法选择基因的可靠性，但扩展面板以检测其他基因将很简单。由于我们能够检测到质粒衍生的序列，我们预计也可以很容易地检测到具有操纵连接、启动子区域和质粒或病毒衍生序列的基因。