Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests

In this 16th edition of the annual banned‐substance review on analytical approaches in human sports drug testing, literature on recent developments in this particular section of global anti‐doping efforts that was published between October 2022 and September 2023 is summarized and discussed. Most recent additions to the continuously growing portfolio of doping control analytical approaches and investigations into analytical challenges in the context of adverse analytical findings are presented, taking into account existing as well as emerging challenges in anti‐doping, with specific focus on substances and methods of doping recognized in the World Anti‐Doping Agency's 2023 Prohibited List. As in previous years, focus is put particularly on new or enhanced analytical options in human doping controls, appreciating the exigence and core mission of anti‐doping and, equally, the conflict arising from the opposingly trending extent of the athlete's exposome and the sensitivity of instruments nowadays commonly available in anti‐doping laboratories.

Section: Discussionmentioning

confidence: 99%

Annual banned‐substance review 16^th edition—Analytical approaches in human sports drug testing 2022/2023

Thevis,

Kuuranne,

Geyer

2023

Gene doping detection in the era of genomics

Li,

Su,

Shi

et al. 2024

Recent progress in gene editing has enabled development of gene therapies for many genetic diseases, but also made gene doping an emerging risk in sports and competitions. By delivery of exogenous transgenes into human body, gene doping not only challenges competition fairness but also places health risk on athletes. World Anti‐Doping Agency (WADA) has clearly inhibited the use of gene and cell doping in sports, and many techniques have been developed for gene doping detection. In this review, we will summarize the main tools for gene doping detection at present, highlight the main challenges for current tools, and elaborate future utilizations of high‐throughput sequencing for unbiased, sensitive, economic and large‐scale gene doping detections. Quantitative real‐time PCR assays are the widely used detection methods at present, which are useful for detection of known targets but are vulnerable to codon optimization at exon–exon junction sites of the transgenes. High‐throughput sequencing has become a powerful tool for various applications in life and health research, and the era of genomics has made it possible for sensitive and large‐scale gene doping detections. Non‐biased genomic profiling could efficiently detect new doping targets, and low‐input genomics amplification and long‐read third‐generation sequencing also have application potentials for more efficient and straightforward gene doping detection. By closely monitoring scientific advancements in gene editing and sport genetics, high‐throughput sequencing could play a more and more important role in gene detection and hopefully contribute to doping‐free sports in the future.

A method for detecting gene doping in horse sports without DNA extraction

Furukawa,

Tozaki,

Kikuchi

et al. 2024

Gene doping is prohibited in horse sports and can involve the administration of exogenous genes, called transgenes, to postnatal animals. Quantitative polymerase chain reaction (qPCR) methods have been developed to detect gene doping; however, these generally require DNA extraction from the plasma prior to qPCR. In this study, we developed two methods, direct droplet digital PCR (ddPCR) and nested ddPCR, to detect the equine erythropoietin (EPO) transgene without DNA extraction. Direct ddPCR used pretreated plasma and PCR to detect the EPO transgene spiked at 10 copies/μL. Nested ddPCR utilised pre‐amplification using nontreated plasma, purification of PCR products and PCR to detect the EPO transgene spiked at 1 copy/μL in plasma. These methods successfully detected the EPO transgene after intramuscular injection into horses. Since each method has different detection sensitivity, the combined use of direct ddPCR for screening and nested ddPCR for confirmation may complement each other and prevent the occurrence of false positives, allowing the reliable detection of gene‐doped substances. One advantage of these methods is the small amount of sample required, approximately 2.2–5.0 μl, owing to the lack of a DNA extraction step. Therefore, these tests could be applied to small volume samples as an alternative to conventional gene doping tests.