title: Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event.
abstract: In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement must be performed. Moreover, for some reference genes no sufficient information on copy number in and among genomes of different lines is available, making adequate quantification difficult. Once developed, the method was subsequently validated according to IUPAC and ISO 5725 guidelines. Thirteen laboratories from 8 EU countries participated in the trial. Eleven laboratories provided results complying with the predefined study requirements. Repeatability (RSDr) values ranged from 8.7 to 15.9%, with a mean value of 12%. Reproducibility (RSDR) values ranged from 16.3 to 25.5%, with a mean value of 21%. Following Codex Alimentarius Committee guidelines, both the limits of detection and quantitation were determined to be <0.1%.
authors.affiliation: European Commission, Joint Research Centre, Institute for Health and Consumer Protection (JRC-IHCP), Via Enrico Fermi, 1, I-21020 Ispra (VA), Italy.
authors.forename: Florian
authors.initials: F
authors.lastname: Weighardt
authors.forename: Cristina
authors.initials: C
authors.lastname: Barbati
authors.forename: Claudia
authors.initials: C
authors.lastname: Paoletti
authors.forename: Maddalena
authors.initials: M
authors.lastname: Querci
authors.forename: Simon
authors.initials: S
authors.lastname: Kay
authors.forename: Marc
authors.initials: M
authors.lastname: De Beuckeleer
authors.forename: Guy
authors.initials: G
authors.lastname: Van den Eede
mesh_terms: D001483:Base Sequence; D002021:Buffers; D002138:Calibration; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D005062:European Union; D005515:Food Labeling; D017343:Genes, Plant / Q000235:genetics*; D018745:Genome, Plant; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D003313:Zea mays / Q000235:genetics*
chemical_list: D002021:Buffers; D018744:DNA, Plant