University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, the Netherlands.
University of Groningen, Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, Groningen, the Netherlands.
Gene Ther. 2019 Aug;26(7-8):338-346. doi: 10.1038/s41434-019-0091-6. Epub 2019 Jul 11.
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.
基因兴奋剂会给运动员带来健康风险，对体育比赛的公平性构成威胁。因此，反兴奋剂组织一直关注基因兴奋剂的检测。以前发表的基于聚合酶链反应的基因兴奋剂检测方法针对的是无内含子转基因的外显子-外显子接头。然而，由于这些接头是已知的，通过篡改 cDNA 序列来逃避检测相对容易。我们已经开发了一种针对潜在兴奋剂基因 EPO、IGF1、IGF2、GH1 和 GH2 的所有外显子-外显子接头的靶向下一代测序检测方法，该方法不易被篡改。使用该检测方法，在 1000ng 基因组 DNA 中，可以检测到 1296 个 cDNA 拷贝的兴奋剂基因的所有外显子-外显子接头，检测灵敏度为 1296 个 cDNA 拷贝。此外，启动子区域和质粒衍生序列在我们的序列数据中很容易检测到。虽然我们展示了我们的方法对一系列基因的可靠性，但将检测面板扩展到检测其他基因将非常简单。由于我们能够检测到质粒衍生序列，我们预计经过接头、启动子区域以及质粒或病毒衍生序列修饰的基因也将很容易被检测到。
