Development and Validation of Two Novel Soybean Endogenous Reference Assays for Qualitative and Quantitative PCR Detection Methods.

Genetically modified (GM) soybean transgenic lines have been approved for commercialization and extensively cultivated worldwide. Event-specific polymerase chain reaction (PCR)-based methods have proven to be one of the most powerful techniques for the detection and quantification of GM organisms (GMOs). To monitor the success of the event-specific PCR-based detection methods, it is essential to include an internal control, which is normally represented by a species-specific endogenous, or reference, gene. Currently, regulatory agencies of countries such as China, Korea, Japan, and EU, have imposed very stringent requirements to PCR conditions of the detection methods. Publically available soybean internal control systems do not always perform robustly under those requirements. In this study, we report the development and validation of two new soybean (Glycine max) internal control assays, gmgba2 and gmgba6, using qualitative gel-based and quantitative real-time (RT)-PCR methods. Both gmgba2 and gmgba6 were specific to soybean and no amplicons were observed and no fluorescent signal was detected in other species. The detection limit of the two assays was as low as 0.012 ng of genomic DNA for qualitative PCR and 0.010 ng of genomic DNA for quantitative PCR methods. Southern blot analysis showed that these two assays are single copy sequences in soybean. Furthermore, biplex qualitative and RT-quantitative PCR methods were designed employing these internal control assays to detect and quantify an herbicide resistant soybean event. These results indicate that the gmgba2 and gmgba6 can be used as internal controls for both qualitative and quantitative PCR detection of GM soybeans.