Multiplex PCR-Liquid Chromatography High-Resolution Tandem Mass Spectrometry for Simultaneous Detection of Multiple Transgenes in Equine Plasma.

This research focuses on a new analytical method to identify multiple doping transgenes in horse plasma. It uses a combination of a multiplex Polymerase Chain Reaction (PCR) and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) for efficient detection.

Introduction to Gene Doping and Methodology

• The study is centered on the rising potential threat of gene doping in sports, which jeopardizes fair play and discredits the integrity of sporting competitions.
• The researchers developed a strategy, utilizing a multiplex PCR process combined with liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS), that allows for the simultaneous identification of multiple transgenes in horse plasma.
• The whole process is carried out within a single reaction, enhancing the efficiency of the testing.

Focus on Specific Transgenes

• The method targets three specific doping transgenes: equine growth hormone 1 (eGH1), equine growth hormone-releasing hormone (eGHRH), and equine interleukin 10 (eIL10).
• An artificial internal control (AIC) is incorporated to be coextracted, coamplified, and codetected with the transgenes, thus minimizing the risk of false negatives.

Design and Optimization Strategies

• The researchers proposed design and optimization strategies for this multiplex PCR-LC-MS approach to achieve high sensitivity in detection.
• The estimated limits of detection were around 25 copies/mL, with estimated limits of confirmation at 50 copies/mL for all targets.

Successful Application and Implications

• The proposed method has been validated and successfully used to confirm the presence of the eIL10 transgene in horse plasma samples administered with the transgene.
• Utilizing LC-HRMS/MS has allowed for the clear identification of transgenes and given the flexibility to test for multiple doping transgenes.
• This setup provides an alternative approach for sensitive, reliable, and multiplex detection of transgenes in most doping control laboratories.