Evaluation of modified PCR quantitation of genetically modified maize and soybean using reference molecules: interlaboratory study.
Real-time polymerase chain reaction (PCR)-based quantitative methods were previously developed and validated for genetically modified (GM) maize or soy. In this study, the quantification step of the validated methods was modified, and an interlaboratory study was conducted. The modification included the introduction of the PCR system SSIIb 3 instead of SSIIb 1 for the detection of the taxon-specific sequence of maize, as well as the adoption of colE1 as a carrier included in a reference plasmid solution as a replacement for salmon testis. The interlaboratory study was conducted with the ABI PRISM 7700 and consisted of 2 separate stages: (1) the measurement of conversion factor (Cf) value, which is the ratio of recombinant DNA (r-DNA) sequence to taxon-specific sequence in each genuine GM seed, and (2) the quantification of blind samples. Additionally, Cf values of other instruments, such as the ABI PRISM 7900 and the ABI PRISM 7000, were measured in a multilaboratory trial. After outlier laboratories were eliminated, the repeatability and reproducibility for 5.0% samples were <15.8 and 20.6%, respectively. The quantitation limits of these methods were 0.5% for Bt11, T25, and MON810, and 0.1% for GA21, Event176, and RR soy. The quantitation limits, trueness, and precision of the current modified methods were equivalent to those of the previous methods. Therefore, it was concluded that the modified methods would be a suitable replacement for the validated methods.
Kodama
Takashi
T
Food and Agricultural Materials Inspection Center, 2-1 Shintoshin, Chuo-ku, Saitama-shi, Saitama 330-9731, Japan.
Kuribara
Hideo
H
Minegishi
Yasutaka
Y
Futo
Satoshi
S
Watai
Masatoshi
M
Sawada
Chihiro
C
Watanabe
Takahiro
T
Akiyama
Hiroshi
H
Maitani
Tamio
T
Teshima
Reiko
R
Furui
Satoshi
S
Hino
Akihiro
A
Kitta
Kazumi
K
D001483:Base Sequence; D004251:DNA Transposable Elements; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification / Q000592:standards; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D013025:Glycine max / Q000235:genetics*; D003313:Zea mays / Q000235:genetics*
D004251:DNA Transposable Elements; D018744:DNA, Plant