Robustness of Digital PCR and Real-Time PCR in Transgene Detection for Gene-Doping Control

Area of Science:

• Sports Science
• Molecular Biology
• Biotechnology

Background:

• Gene doping, the use of exogenous genes (transgenes) for performance enhancement, is prohibited in sports.
• Quantitative PCR (qPCR) methods are employed for transgene detection, crucial for anti-doping regulations.
• The reliability of qPCR methods can be challenged by variations in primer and probe sequences.

Purpose of the Study:

• To evaluate the robustness of digital PCR (dPCR) and real-time PCR (qPCR) in detecting transgenes.
• To assess the impact of mismatched primers and probes on the sensitivity and accuracy of these PCR methods.
• To determine optimal conditions for enhancing transgene detection in anti-doping scenarios.

Main Methods:

• Comparison of dPCR and qPCR assays using matched (P-true) and mismatched (P-false) primers and probes against template DNA.
• Analysis of fluorescence intensity, copy number (dPCR), and quantification cycle (Cq) values (qPCR) under varying conditions.
• Investigation of the effect of reduced annealing temperature on PCR performance with mismatched sequences.

Main Results:

• Mismatched probes significantly reduced fluorescence intensity in both dPCR and qPCR.
• dPCR provided consistent copy number results irrespective of probe matching, while qPCR showed reduced sensitivity (higher Cq values) with mismatched probes.
• Lowering the annealing temperature improved dPCR copy number detection, even with mismatched probes, indicating enhanced robustness.

Conclusions:

• Digital PCR demonstrates superior robustness compared to real-time PCR when detecting transgenes with sequence variations.
• Optimizing PCR conditions, such as lowering annealing temperature, can improve sensitivity and mitigate false negatives in transgene detection.
• Digital PCR is a more reliable method for detecting potential gene doping, especially when athletes attempt to evade detection through sequence modification.