Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products

An event-specific PCR method for detection and quantification of geneticall y modified Roundup Ready soybean (RRS) is described in this article. The co mplete DNA sequence at both the right and left integration sites of this ge netically modified organism has recently been determined. Based on these se quence data, transformation event-specific primer pairs were developed. The se primers amplify a fragment of the unique junction region between the ins erted DNA and the plant DNA and therefore act as unique identifiers. Two se nsitive, qualitative PCR assays gave absolute detection limits of 5 copies of the RRS junction fragment in 100 pg of total DNA per reaction. A real-ti me PCR method was then developed with the LightCycler System. For determina tion of the RRS content, a completely new type of external calibration stan dard is introduced here. A fragment of the RRS specific junction region and a fragment of the endogenous soybean lectin gene were both cloned in a pla smid vector. These new diagnostic DNA fragments allow quantification of RRS in whichever type of matrix, in a range of 10-10(6) copies of each target.