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Direct LC-HRMS/MS characterisation and RT-qPCR detection for potential mRNA-based doping agent.

Bruce P-N Yuen1, Kin-Sing Wong2, Venus Y-C Lin2

  • 1Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China.

Analytical and Bioanalytical Chemistry
|May 1, 2026
PubMed
Summary
This summary is machine-generated.

Researchers developed a method to detect engineered erythropoietin (EPO) messenger RNA (mRNA) for equine anti-doping. This mass spectrometry and RT-qPCR assay verifies mRNA identity and detects doping agents in sports.

Keywords:
Equine sportsGene doping detectionLC–MSMappingRT–qPCRmRNA therapeutics

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Sports Science

Background:

  • Messenger ribonucleic acid (mRNA) therapeutics show promise for protein expression.
  • Potential misuse of mRNA for gene doping in sports presents an emerging risk.
  • Detection of modified mRNA, like erythropoietin (EPO) mRNA, is crucial for anti-doping efforts.

Purpose of the Study:

  • To verify the identity and chemical composition of an online-available human EPO mRNA product.
  • To develop a sensitive detection assay for equine anti-doping.
  • To establish a framework for identifying novel mRNA-based doping agents.

Main Methods:

  • Mass spectrometry (MS)-based bottom-up workflow with RNase 4 digestion and LC-MS/MS analysis.
  • Automated sequence mapping against an mRNA database for identity verification.
  • Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay development for equine plasma.

Main Results:

  • Successful verification of mRNA sequence and 5-methoxyuridine base modification using MS.
  • Achieved mean sequence coverage above 74% for product identity confirmation.
  • Validated RT-qPCR assay with a limit of detection at 1250 copies/mL of EPO mRNA in equine plasma.

Conclusions:

  • The combined MS and RT-qPCR approach effectively verifies mRNA identity and composition.
  • The developed assay provides a reliable method for detecting EPO mRNA in equine anti-doping.
  • This framework can be extended to monitor other emerging mRNA-based doping agents in sports.