Quantitative determination of genetic modification markers in tobacco by high-throughput real time PCR
Analytical methods based on the polymerase chain reaction (PCR) are widely used to detect, identify and quantify genetically modified organisms (GMOs) in agricultural crops, food and feed. The determination of genetic identity facilitates the implementation of identity preservation (IP) guidelines which aim at preserving the integrity and quality of crop products through traceability along the whole supply chain. We have developed a quantitative method based on real time PCR which can detect the presence and accurately measure the amount of GM materials in different tobacco matrices, i.e. in seed, seedlings, green leaf, cured and processed leaf. This method employs the ABI PRISM® 7700 platform and dual-labeled fluorogenic probes (TaqMan® probes) to detect two DNA sequences that are frequently used in plant transformation: the cauliflower mosaic virus 35S promoter and the Agrobacterium tumefaciens nos terminator. GMO quantitation (in % w/w of the total tobacco material) is accomplished by calibration with GM tobacco reference materials, including the normalization for variations in DNA concentrations between analyses. The target sequences are co-amplified with an endogenous tobacco reference gene in a multiplex reaction. Unlike conventional PCR techniques, real time PCR does not require post-PCR sample handling, thus minimizing the risk of carry-over contamination and resulting in higher throughput assays. This paper will review the state-of-the-art of PCR-based quantitative determination of GMOs and will describe its applications for quantification of GM tobacco.