Multiplex real-time PCR for the simultaneous detection and quantification of DNA from three transgenic rice species and construction and application of an artificial oligonucleotide as reference molecule

According to the EU and Swiss legislation for food, only approved traits of transgene plants are allowed to be imported and sold to the consumer. In order to control imports of rice and rice products from retailers, dealers and importing companies, efficient and reliable methods for the detection and quantification are a prerequisite. Therefore, a novel pentaplex real-time polymerase chain reaction system was developed and validated for the quantitative determination of three genetically modified rice lines at once. This system simultaneously determines DNA contents of the phospholipase-gen, a rice species specific gene, the 35S:BAR-construct, as the promotor of different transgene rice lines and the specific systems for LL62, LL601 and Bt-63-rice (Shanyou63). The test exhibits a good specificity and sensitivity for the transgenes in the range of 0.01-1%. It proved its efficiency and reliability in daily routine. Due to the lack of appropriate reference material for the Bt-63-rice, a reference oligonucleotide was artificially constructed. This oligonucleotide proved its applicability in diagnostic analysis.