Event-specific qualitative and quantitative PCR detection of roundup ready event GT73 based on the 3′-integration junction

Abstract

With the development of genetically modified organisms, labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed state-of-art. This paper describes an event-specific PCR method for qualitative and quantitative of Roundup Ready canola event GT73. The 3'-integration junction was characterized by two methods: inverse-PCR and thermal asymmetric interlaced-PCR. In the conventional qualitative PCR assay, the event-specific primers designed were confirmed to be specific and the limit of detection (LOD) was 0.05% (approximates to ten haploid genome copies). In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were five and ten haploid genome copies, respectively. In addition, for further quantitative detection, a reference molecule which contained the canola endogenous gene and event-specific sequence was constructed and standard curves were set up. The goodness of the linearity and high efficiency of the PCR reaction indicated the usability of the plasmid and the established PCR system. Moreover, mixed samples with different GT73 content (6, 3, 1 and 0.5%) were quantified using the established real-time PCR system to evaluate the trueness and precision of the system. The trueness expressed as bias varied from 2.00 to 18.00%. The precision expressed as variation coefficient were different from 6.40 to 32.95%. From above results, we believed that the established event-specific qualitative and quantitative PCR systems for GT73 in this study were acceptable and suitable for genetic modified canola detection.