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

Abstract.

An event-specific PCR method for detection and quantification of genetically modified Roundup Ready soybean (RRS) is described in this article. The complete DNA sequence at both the right and left integration sites of this genetically modified organism has recently been determined. Based on these sequence data, transformation event-specific primer pairs were developed. These primers amplify a fragment of the unique junction region between the inserted DNA and the plant DNA and therefore act as unique identifiers. Two sensitive, 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-time PCR method was then developed with the LightCycler System. For determination of the RRS content, a completely new type of external calibration standard 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 plasmid vector. These new diagnostic DNA fragments allow quantification of RRS in whichever type of matrix, in a range of 10–10⁶ copies of each target.