Development of taxon-specific sequences of common wheat for the detection of genetically modified wheat.
Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.
Iida
Mayu
M
QE Center, Research and Development, Quality Assurance Division, Nisshin Seifun Group Inc., 5-3-1 Tsurugaoka, Saitama 356-8511, Japan.
Yamashiro
Satomi
S
Yamakawa
Hirohito
H
Hayakawa
Katsuyuki
K
Kuribara
Hideo
H
Kodama
Takashi
T
Furui
Satoshi
S
Akiyama
Hiroshi
H
Maitani
Tamio
T
Hino
Akihiro
A
D001483:Base Sequence; D015139:Blotting, Southern; D018744:DNA, Plant / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012639:Seeds / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014908:Triticum / Q000235:genetics*
D018744:DNA, Plant