Development of a duplex chamber digital PCR to quantify twelve genetically modified maize events

Area of Science:

• Agricultural Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Accurate quantification of genetically modified organisms (GMOs) is crucial for regulatory compliance, particularly with threshold-based labeling policies.
• Existing methods like real-time quantitative PCR (qPCR) require standard curves and can be less efficient.
• South Korea has specific labeling thresholds, such as the 3.0% rule, necessitating precise GMO detection methods.

Purpose of the Study:

• To develop and validate event-specific duplex chamber/chip-based digital PCR (cdPCR) methods for quantifying approved GM maize events in South Korea.
• To assess the accuracy, sensitivity, and robustness of cdPCR compared to conventional qPCR for GMO analysis.
• To evaluate the suitability of cdPCR for regulatory applications and enforcement of labeling thresholds.

Main Methods:

• Development and validation of twelve event-specific duplex cdPCR assays using microfluidic array plates.
• Absolute quantification of GM maize events at 0.9%, 3.0%, and 5.0% GMO content levels.
• Incorporation of event-specific zygosity ratio correction for enhanced measurement accuracy.
• Comparative performance analysis against real-time quantitative PCR (qPCR).

Main Results:

• The validated cdPCR methods demonstrated high sensitivity, robustness, trueness, precision, and reproducibility, meeting international performance criteria.
• cdPCR enabled absolute quantification without standard curve calibration, simplifying the process.
• Comparative analysis showed cdPCR accuracy was comparable to qPCR, with added benefits of cost-efficiency and operational simplicity.
• The methods proved effective for quantifying GM maize events at various levels, including those relevant to South Korea's 3.0% labeling threshold.

Conclusions:

• Duplex cdPCR is a practical and reliable tool for accurate GMO quantification.
• The developed cdPCR methods are suitable for national regulatory laboratories and the enforcement of GMO labeling thresholds.
• cdPCR offers advantages over qPCR in terms of cost and operational simplicity for routine GMO analysis.