A screening method for the detection of the 35S promoter and the nopaline synthase terminator in genetically modified organisms in a real-time multiplex polymerase chain reaction using high-resolution melting-curve analysis.
To screen for unauthorized genetically modified organisms (GMO) in the various crops, we developed a multiplex real-time polymerase chain reaction high-resolution melting-curve analysis method for the simultaneous qualitative detection of 35S promoter sequence of cauliflower mosaic virus (35SP) and the nopaline synthase terminator (NOST) in several crops. We selected suitable primer sets for the simultaneous detection of 35SP and NOST and designed the primer set for the detection of spiked ColE1 plasmid to evaluate the validity of the polymerase chain reaction (PCR) analyses. In addition, we optimized the multiplex PCR conditions using the designed primer sets and EvaGreen as an intercalating dye. The contamination of unauthorized GMO with single copy similar to NK603 maize can be detected as low as 0.1% in a maize sample. Furthermore, we showed that the present method would be applicable in identifying GMO in various crops and foods like authorized GM soybean, authorized GM potato, the biscuit which is contaminated with GM soybeans and the rice which is contaminated with unauthorized GM rice. We consider this method to be a simple and reliable assay for screening for unauthorized GMO in crops and the processing food products.
Akiyama
Hiroshi
H
National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. akiyama@nihs.go.jp
Nakamura
Fumi
F
Yamada
Chihiro
C
Nakamura
Kosuke
K
Nakajima
Osamu
O
Kawakami
Hiroshi
H
Harikai
Naoki
N
Furui
Satoshi
S
Kitta
Kazumi
K
Teshima
Reiko
R
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D003087:Colicins / Q000235:genetics; D012275:Oryza / Q000235:genetics; D030821:Plants, Genetically Modified; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D013728:Terminator Regions, Genetic
D003087:Colicins; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase