Assessment of the genetic stability of GMOs with a detailed examination of MON810 using Scorpion probes

摘要

For the cultivation and maintenance of seeds, genetic mutations have to be avoided as much as possible. According to the Directive 2001/18/EC, genetic stability has to be proven for the approval of GMOs [1]. The investigation of genetic stability can be performed with various methods, but Southern blots are most commonly used. In this study, a different method, a real-time PCR involving Scorpion primer, was developed for the analysis of DNA rearrangements in GMOs. The advantage of this method is that already small changes in sequence can be detected. In addition, large numbers of samples can be tested at once in a single PCR run. Possible alterations in the region of the Scorpion primer can be detected by differences in the real-time PCR plots. The sequence that was analysed contained 130 nucleotides. The sensitivity of Scorpion primers to measure genetic stability was verified in experiments with plasmids that had only minor sequence alterations in the target region. Such small differences led to a total reduction of the fluorescence signal, even in heterozygous samples where the unaltered template was present as well. Scorpion primers were then used to investigate the transgenic sequences of 567 individual seeds of MON810 for maize at the 5′ and the 3′ region of the insert. In combination with SYBR green, the screening procedure allowed a selection of samples for further analysis. If a sample showed low fluorescence with the Scorpion primer but a normal amplification with SYBR green, then the integrity of the insert was checked by sequencing. The results, however, gave no indication of a mutation or rearrangement of MON810 seeds. In additional experiments, the reliability of the method was studied by determining several parameters that are required for a validation, e.g., specificity, cross-reactivity, dynamic range, correlation of the template concentration and the corresponding Ct values, PCR efficiency, relative repeatability standard deviation (RSDr) relative reproducibility standard deviation (RSDR).