Evaluation of modified PCR quantitation of genetically modified maize and soybean using reference molecules:...

Area of Science:

• Agricultural Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Validated real-time quantitative polymerase chain reaction (PCR) methods exist for genetically modified (GM) maize and soy.
• Modifications to existing validated methods were investigated to potentially improve detection and quantification.

Purpose of the Study:

• To modify and validate real-time quantitative PCR methods for GM maize and soy through an interlaboratory study.
• To assess the performance of modified methods using different PCR systems and carriers.

Main Methods:

• Modified the quantification step of validated real-time PCR methods, introducing PCR system SSIIb 3 and colE1 carrier.
• Conducted a two-stage interlaboratory study involving conversion factor (Cf) value measurement and blind sample quantification.
• Measured Cf values on ABI PRISM 7700, 7900, and 7000 instruments.

Main Results:

• Repeatability and reproducibility for 5.0% samples were <15.8% and 20.6%, respectively, after outlier elimination.
• Quantitation limits were 0.5% for Bt11, T25, MON810 (maize) and 0.1% for GA21, Event176, RR soy.
• Modified methods demonstrated equivalent quantitation limits, trueness, and precision compared to previous validated methods.

Conclusions:

• The modified real-time quantitative PCR methods are suitable replacements for previously validated methods.
• The study confirmed the reliability and accuracy of the enhanced methods for GM crop analysis.