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Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control.

Teruaki Tozaki¹, Aoi Ohnuma¹, Natasha A Hamilton²

¹Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi, Japan.

Drug Testing and Analysis

|October 5, 2021

Summary

A new nested digital polymerase chain reaction (PCR) method reliably detects low-copy transgenes, crucial for preventing gene doping in sports. This technique enhances accuracy, reducing false negatives and positives in anti-doping tests.

Keywords:

digital PCR gene doping horseracing transgene

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Area of Science:

Sports Science
Molecular Biology
Biotechnology

Background:

Gene doping, the use of transgenes in athletes, undermines fair competition in human and horse sports.
Current transgene detection methods, including quantitative PCR, struggle to reliably identify low-level gene doping instances.
Accurate detection of low-copy transgenes is essential for effective anti-doping enforcement.

Purpose of the Study:

To develop and validate a highly sensitive method for detecting low-copy transgenes.
To improve the reliability of gene doping detection in sports.
To minimize false positives and false negatives in anti-doping analyses.

Main Methods:

Development of a nested digital PCR assay combining conventional PCR preamplification with droplet digital PCR detection.
Validation of the method using varying low concentrations of transgene templates (5-120 copies).
Assessment of the impact of common inhibitors and variations in primers/probes on assay performance.

Main Results:

The nested digital PCR method successfully detected low-copy transgenes with high sensitivity.
Quantification of transgene copies from 5 to 120 copies per template demonstrated assay linearity.
The method showed limited inhibition by common biological and chemical substances, and robustness against minor primer/probe modifications.

Conclusions:

The developed nested digital PCR is a powerful tool for detecting low-copy transgenes.
This method significantly enhances the accuracy of gene doping detection in sports.
It offers a reliable qualitative test to prevent false negatives and false positives in anti-doping efforts.