Gene doping involves the misuse of genetic materials to alter an athlete’s performance, which is banned at all times in both human and equine sports. Quantitative polymerase chain reaction (qPCR) assays have been used to control the misuse of transgenes in equine sports. Our laboratory recently developed and implemented duplex as well as multiplex qPCR assays for transgenes detection. To further advance gene doping control, we have developed for the first time a sensitive and definitive PCR-liquid chromatography high-resolution tandem mass spectrometry (PCR-LC-HRMS/MS) method for transgene detection with an estimated limit of detection of below 100 copies/mL for the human erythropoietin (hEPO) transgene in equine plasma. The method involved magnetic-glass-particle-based extraction of DNA from equine plasma prior to PCR amplification with 2′-deoxyuridine 5′-triphosphate (dUTP) followed by treatments with uracil DNA glycosylase and hot piperidine for selective cleavage to give small oligonucleotide fragments. The resulting DNA fragments were then analyzed by LC-HRMS/MS. The applicability of this method has been demonstrated by the successful detection of hEPO transgene in a blood sample collected from a gelding (castrated male horse) that had been administered the transgene. This novel approach not only serves as a complementary method for transgene detection but also paves the way for developing a generic PCR-LC-HRMS/MS method for the detection of multiple transgenes.

中文翻译：

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通过 PCR-液相色谱高分辨率串联质谱法对马血浆中的人促红细胞生成素转基因进行基因掺杂控制分析

基因兴奋剂涉及滥用遗传物质来改变运动员的表现，这在人类和马术运动中都是被禁止的。定量聚合酶链反应（qPCR）检测已被用来控制马术运动中转基因的滥用。我们的实验室最近开发并实施了用于转基因检测的双重和多重 qPCR 测定。为了进一步推进基因兴奋剂控制，我们首次开发了一种灵敏且确定的PCR-液相色谱高分辨率串联质谱（PCR-LC-HRMS/MS）转基因检测方法，估计检测限低于100马血浆中人促红细胞生成素 (hEPO) 转基因的拷贝数/mL。该方法包括利用磁玻璃颗粒从马血浆中提取 DNA，然后用 2'-脱氧尿苷 5'-三磷酸 (dUTP) 进行 PCR 扩增，然后用尿嘧啶 DNA 糖基化酶和热哌啶处理进行选择性切割，得到小的寡核苷酸片段。然后通过 LC-HRMS/MS 分析所得 DNA 片段。该方法的适用性已通过从已施用转基因的骟马（阉割的雄性马）收集的血液样本中成功检测到 hEPO 转基因而得到证明。这种新方法不仅可以作为转基因检测的补充方法，而且还为开发用于检测多种转基因的通用PCR-LC-HRMS/MS方法铺平了道路。