Detection of eight genetically modified canola events using two event-specific pentaplex PCR systems

An event-specific multiplex polymerase chain reaction (PCR) detection method was developed to simultaneously detect eight genetically modified (GM) canola events (GT73, MS1, MS8, RF1, RF2, RF3, T45, and Topas 19/2). For a successful multiplex PCR assay, the eight GM canola events were divided into groups 1 and 2 in consideration of their amplicon sizes, primer efficiencies, and target sequences. In addition to the canola endogenous reference gene, FatA, the two pentaplex PCR assays targeted group 1, containing GT73, MS8, RF3, and T45, and group 2, including MS1, RF1, RF2, and Topas 19/2. Event-specific primers targeting the eight GM canola events were designed, and their specificities were confirmed using 14 GM events of maize, soybean, cotton, and canola. After optimizing the reaction conditions, the limits of detection of these two assays were approximately 0.025% for group 1 and 0.0125% for group 2. This multiplex PCR method for eight GM canola events was validated by two operators, and the data confirmed the reliability of the developed assays. The method was used to test commercially available canola seed (eight samples) and meal (one sample) produced in South Korea, China, Canada, and Australia, and the results revealed one or more GM canola events in seven of the nine samples tested. These results show that the developed multiplex system is applicable for use in the specific testing of eight commercially available GM canola events.