Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan.
Equine Department, Japan Racing Association, 6-11-1 Roppongi, Minato, Tokyo 106-8401, Japan.
Genes (Basel). 2020 Apr 23;11(4):457. doi: 10.3390/genes11040457.
Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/μL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.
基因兴奋剂是一种滥用和误用基因治疗的行为，是体育和赛马行业的主要关注点。迫切需要能够检测和监测基因兴奋剂的有效方法。虽然已经开发出几种基于 PCR 的检测转基因的方法，但其中许多方法仅针对单个转基因。然而，许多与运动能力相关的基因可能是潜在的基因兴奋剂物质。在这里，我们开发了一种针对多个转基因的检测方法。我们针对可能与运动表现相关的 12 个基因，并为每个基因设计了两个 TaqMan 探针/引物对。准备了一个包含 24 个检测的面板，并通过使用集成流控 (IFC) 的微流控定量 PCR (MFQPCR) 系统进行检测。该面板的检测限为 6.25 拷贝/μL。使用几种浓度的参考材料和动物基因组 DNA 验证了扩增特异性，导致特异性检测。此外，通过 MFQPCR 成功地在一匹注射了 20 毫克转基因的马中实现了靶标特异性检测。因此，MFQPCR 可以被认为是基因兴奋剂控制中多目标检测的一种合适方法。据我们所知，这是微流控 qPCR (MFQPCR) 首次应用于赛马基因兴奋剂控制。
