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.
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.
Corbisier
Philippe
P
European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, 2440 Geel, Belgium. philippe.corbisier@ec.europa.edu.
Broothaerts
Wim
W
Gioria
Sabrina
S
Schimmel
Heinz
H
Burns
Malcolm
M
Baoutina
Anna
A
Emslie
Kerry R
KR
Furui
Satoshi
S
Kurosawa
Yasunori
Y
Holden
Marcia J
MJ
Kim
Hyong-Ha
HH
Lee
Yun-Mi
YM
Kawaharasaki
Mamoru
M
Sin
Della
D
Wang
Jing
J
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*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018744:DNA, Plant; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis