Development and applications of real-time PCR standards for GMO quantification based on tandem-marker plasmids

The need to comply with mandatory thresholds introduced for the labeling of GM food and feed by recent European Regulations is increasing the interest for reliable standards for GMO quantification. Certified reference materials (CRMs) for the different GMO lines are an essential element to calibrate measurement systems adopted to quantify GM ingredients. Unfortunately, conventional CRMs are affected not only by their high production costs but also by time consuming production and certification procedures. At the moment CRMs are available only for few of the GMOs authorized in Europe. In addition, latest developments in European legislation makes conceivable that the number of different authorized GM lines to be analysed will increase significantly, raising the need for additional CRMs. In the field of GMO detection and quantification, plasmids have been demonstrated to represent a cheap and reliable alternative to conventional CRMs. Here we describe the production of tandem-marker plasmids containing simultaneously specific target sequences for the GMO of interest and a species-specific reference marker sequence in a 1:1 ratio have been developed and used as real-time PCR standards. Strengths and weaknesses of these novel standards are discussed in detail.