title: Toward metrological traceability for DNA fragment ratios in GM quantification. 1. Effect of DNA extraction methods on the quantitative determination of Bt176 corn by real-time PCR.
abstract: An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.
authors.affiliation: European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, 2440 Geel, Belgium. philippe.corbisier@ec.europa.edu.
authors.forename: Philippe
authors.initials: P
authors.lastname: Corbisier
authors.forename: Wim
authors.initials: W
authors.lastname: Broothaerts
authors.forename: Sabrina
authors.initials: S
authors.lastname: Gioria
authors.forename: Heinz
authors.initials: H
authors.lastname: Schimmel
authors.forename: Malcolm
authors.initials: M
authors.lastname: Burns
authors.forename: Anna
authors.initials: A
authors.lastname: Baoutina
authors.forename: Kerry R
authors.initials: KR
authors.lastname: Emslie
authors.forename: Satoshi
authors.initials: S
authors.lastname: Furui
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Kurosawa
authors.forename: Marcia J
authors.initials: MJ
authors.lastname: Holden
authors.forename: Hyong-Ha
authors.initials: HH
authors.lastname: Kim
authors.forename: Yun-Mi
authors.initials: YM
authors.lastname: Lee
authors.forename: Mamoru
authors.initials: M
authors.lastname: Kawaharasaki
authors.forename: Della
authors.initials: D
authors.lastname: Sin
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
mesh_terms: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D018744:DNA, Plant / Q000032:analysis / Q000302:isolation & purification*; D004731:Endotoxins / Q000235:genetics*; D030841:Food, Genetically Modified; 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; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis