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
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概要
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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.
著者
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TESHIMA Reiko
National Institute of Health Sciences
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AKIYAMA Hiroshi
National Institute of Health Sciences
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KAWAKAMI HIROSHI
Department of Gastroenterology, Hokkaido University Graduate School of Medicine
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KITTA Kazumi
National Food Research Institute
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Nakajima Osamu
National Inst. Of Health Sciences
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Harikai Naoki
School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University
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Nakamura Kosuke
National Institute of Health Sciences
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Yamada Chihiro
Department of Food Science & Nutrition, Kyoritsu Womens University
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Nakamura Fumi
National Institute of Health Sciences
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Furui Satoshi
National Food Research Institute
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Kitta Kazumi
National Food Res. Inst.
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Teshima Reiko
National Inst. Of Health Sciences
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Harikai Naoki
School Of Pharmaceutical Sciences Mukogawa Women's University
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Furui Satoshi
National Food Res. Inst.
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Akiyama Hiroshi
National Food Research Institute National Agriculture And Food Research Organization
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Kawakami Hiroshi
Department Of Gastroenterology And Hepatology Hokkaido University
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Harikai Naoki
School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University
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Kawakami Hiroshi
Department of Food Science & Nutrition, Kyoritsu Women's University
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Kawakami Hiroshi
Department of Cardiology, Ehime Prefectural Central Hospital
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