Validation of different genomic and cloned DNA calibration standards for construct-specific quantification of LibertyLink in rapeseed by real-time PCR

Abstract

A construct-specific real-time PCR assay was developed for quantitative detection of LibertyLink in rapeseed by using the junction region between the 35S promoter and the pat gene. For validation of precision and accuracy of quantification five external genomic and cloned DNA calibration standards were constructed. Seeds of non-transgenic rapeseed were pooled with appropriate numbers of seeds of transgenic rapeseed. Genomic DNA from transgenic rapeseed was diluted with corresponding volumes of water or genomic DNA from non-transgenic rapeseed. Plasmids containing DNA fragments of the construct-specific and a brassica-specific reference gene were diluted with appropriate volumes of water and last, corresponding amounts of plasmids containing DNA fragments of the construct-specific construct were spiked in genomic DNA from non-transgenic rapeseed. The approximately estimated upper and lower limits of the quantification of calibration standards constructed with mixed seeds, plasmids in genomic background and water-diluted genomic DNA of 206–279 and 21–28 copy numbers were similar, whereas water-diluted plasmid and mixed genomic DNA standards never reached the quantification thresholds of 13 and 26 copy numbers, respectively. The accuracy of quantification for all calibration standards was tested by calculating the recovery of quantification for reference samples. The highest accuracy was obtained by using standards of mixed genomic DNA and plasmids in genomic background. This indicates a significant influence of background DNA on the quantification of genetically modified (GM) contaminations in rapeseed. Since construct-specific plasmids can be produced on a large scale, stored for a long time without loss of quality and spiked in different DNA backgrounds, plasmid standards can be used in GM quantification as 'gold standards'.