Test kit and method for quantitatively detecting genetically modified DNA in foodstuff by means of fluorescence-coupled PCR

A method of quantitatively detecting genetically modified DNA (transgene) in foodstuffs,

• characterized in that
• the detection is performed using fluorescence-coupled PCR, said method involving extracting the total DNA from the food sample, and
• a) determining the amount of transgene in the total DNA by performing a PCR reaction with a transgene-specific fluorescence-labelled probe S1 and two transgene-specific primers P1 and P2 in a first reaction vessel and measuring the change in fluorescence radiation as compared to a control, a synthetic gene fragment (target IAC DNA) in addition to the primers P1 and P2 and a fluorescence-labelled probe S2 being used as internal amplification control (IAC) for transgene determination in the first reaction vessel, which fragment has two binding sites for the primers P1 and P2 and one binding site for the fluorescence-labelled probe S2 which not only differs from probe S1 in its sequence but also is labelled with a fluorescent dye other than that of probe S1;
• b) selecting a reference gene and determining the amount of reference gene in the total DNA by performing a PCR reaction with a reference gene-specific fluorescence-labelled probe S3 and two reference gene-specific primers P3 and P4 in a second reaction vessel and measuring the change in fluorescence radiation as compared to a control, a synthetic gene fragment (reference IAC DNA) in addition to the primers P3 and P4 and the fluorescence-labelled probe S2 being used as internal amplification control (IAC) for reference gene determination in the second reaction vessel, which fragment has two binding sites for the primers P3 and P4 and one binding site for the fluorescence-labelled probe S2 which is identical to the fluorescence-labelled probe S2 in the target gene system, but differs from probe S3 with respect to sequence and fluorescent dye;
• and
• ultimately, calculating the level of genetically modified DNA from the ratio of the amounts of transgene and reference gene.