title: Toward metrological traceability for DNA fragment ratios in GM quantification. 2. Systematic study of parameters influencing the quantitative determination of MON 810 corn by real-time PCR.
abstract: This paper is part of a set of three papers investigating metrological traceability of the quantification of DNA fragments as, for instance, used for quantification of genetic modifications. This paper evaluates the possible impact of several factors on results of real-time Polymerase Chain Reaction (PCR) measurements. It was found that the particle size of the powder samples does not have an influence, whereas the nature of the calibrant (plasmidic or genomic DNA) has a significant effect. Moreover, two real-time PCR detection methods (construct-specific and event-specific) for MON 810 corn were compared. The results obtained in a specifically designed interlaboratory study revealed a significant influence of the DNA extraction method on measurement results when the MON 810 construct-specific real-time PCR detection method was applied. Statistical analyses confirmed the importance of validating DNA extraction methods in conjunction with real-time PCR methods.
authors.affiliation: European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, 2440 Geel, Belgium. diana.charels@ec.europa.eu
authors.forename: Diana
authors.initials: D
authors.lastname: Charels
authors.forename: Sylvia
authors.initials: S
authors.lastname: Broeders
authors.forename: Philippe
authors.initials: P
authors.lastname: Corbisier
authors.forename: Stefanie
authors.initials: S
authors.lastname: Trapmann
authors.forename: Heinz
authors.initials: H
authors.lastname: Schimmel
authors.forename: Thomas
authors.initials: T
authors.lastname: Linsinger
authors.forename: Hendrik
authors.initials: H
authors.lastname: Emons
mesh_terms: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D018744:DNA, Plant / Q000032:analysis*; D004731:Endotoxins / Q000235:genetics; D030841:Food, Genetically Modified; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012639:Seeds / Q000235:genetics; D003313:Zea mays / Q000235:genetics*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018744:DNA, Plant; D004731:Endotoxins; D018840:HSP70 Heat-Shock Proteins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis