Development and evaluation of qualitative detection methods for unapproved genetically modified rice (LLRice)

We developed a specific method to extract DNA from rice grain samples and modified the qualitative real-time PCR method provided by Bayer Co., Ltd. for reliable detection of the genetically modified (GM) rice variety, LLRice601, which has not undergone safety assessment for regulatory approval in Japan. Moreover, we conducted a data analysis to confirm the results obtained with real-time PCR. The yields of DNA extracted from powdered samples of rice grains were almost equal among 5 different varieties of rice, and there was no significant difference in the yield over three days. Reliable results were obtained using 50 ng of the extracted DNA as the template for real-time PCR. To examine the adequacy of the methods, we organized an interlaboratory study with the participation of 2 laboratories, in which 80 test samples were analyzed in a blinded manner. The statistical analysis revealed no significant difference in the Ct value for the endogenous gene of the DNA samples and for the targeted DNA sequence of 0.1% samples. The limit of detection of the method was approximately 0.1%. Analysis of the fluorescence intensity of the PCR-amplified product of the construct-specific DNA sequence suggested that it may be reasonable to judge a sample as positive when a Ct value of less than 40 is obtained.