Establishment Of Event-Specific Real-Time PCR Detection System For Genetically Modified Soybean MON89788

Genetically modified (GM) glyphosate-tolerant soybean event MON8978 was developed by the Monsanto Co. and exported to China as raw material in 2008. The purpose of study was to establish event-specific real-time PCR detection system for genetically modified soybean MON89788. It will provide the technological and theoretical basis for the regulation of GM soybean MON89788 and its products. The experiment included two parts:The first experiment was designed to screen a suitable DNA extraction method of soybean and its products, which will provide a reference system for comparison and evaluation of DNA extraction methods. In this study, some changes were made with CTAB method, SDS method and Tiangen kit method according to the need of detection, and these methods were used to extract DNA from soybean, soybean milk powder, dried soybean curd, soybean protein and soybean meal. After extraction, the quality of soybean DNA was examined with ultraviolet spectrophotometry and real-time fluorescent PCR, respectively. According to the results, compared with SDS method and Tiangen kit method, CTAB method extracted higher quality DNA which was more able to ensure the accuracy of PCR detection of GM ingredients. Meanwhile, CTAB method had low cost and simple operation. Therefore, among three methods CTAB method is the most suitable to extract DNA from soybean, soybean milk powder, dried soybean curd, soybean protein and soybean meal for detection of GM ingredients.The second experiment was designed to establish an event-specific real-time PCR dedection method for GM soybean MON89788, which will provide the theoretical and technological basis for detection of GM soybean MON89788 and its products. In this study, a new reference molecule pUC19-LM5 containing event-specific sequence of MON89788 soybean and soybean endogenous gene lectin was constructed. An event-specific real-time PCR method for GM soybean MON89788 using pUC19-LM5 as calibrator was established and validated. The established method was highly specific to GM soybean MON89788 detection. The limits of detection (LODs) and limits of quantification (LOQs) were 4 and 10 copies of pUC19-LM5 DNA for both MON89788 event-specific sequence and lectin. Two standard curves for MON89788 event-specific sequence and lectin showed high PCR efficiencies (96% or 95%) and good linearity (R2>0.99). For quantification of three blind samples, low bias (ranging from -14.81% to 9.81%) and relative standard deviations (ranging from 2.72% to 6.28%) were obtained. Moreover, the established method was successfully applied to detection of practical samples. According to the results, the established event-specific real-time PCR method was suitable for identification and quantification of GM soybean MON89788 and its products.