Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products — Part 4: Real-time PCR based screening methods for the detection of the P-nos and P-nos-nptII DNA sequen...
ISO/TS 21569-4:2016 specifies a procedure for the detection of a DNA sequence of the promoter region of the nopaline synthase gene (P-nos) from Agrobacterium tumefaciens and a procedure for the detection of the DNA transition sequence between P-nos and the neomycin-phosphotransferase gene (nptII) from the Tn5 transposon of Escherichia coli K12. The nos-promoter and the P-nos-nptII-construct are frequently found in genetically modified plants. The P-nos and P-nos-nptII specific methods are based on real-time PCR and can be used for qualitative screening purposes. For identification and quantification of a specific genetically modified plant (event) a follow-up analysis has to be carried out. The methods described are applicable for the analysis of DNA extracted from foodstuffs. They may also be suitable for the analysis of DNA extracted from other products such as feedstuffs and seeds. The application of these methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix. The DNA sequence amplified by the P-nos element-specific method can be detected in samples which contain DNA of the naturally occurring Ti-plasmid of A. tumefaciens. For this reason, it is necessary to confirm a positive screening result. Further analyses are required using construct-specific or event specific methods.