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.
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.
复制标题
使用高分辨率熔解曲线分析在实时多重聚合酶链式反应中检测转基因生物体中 35S 启动子和胭脂碱合酶终止子的筛选方法。
DOI：
10.1248/bpb.32.1824
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发表时间：
2009
影响因子：
2
通讯作者：
R. Teshima
中科院分区：
文献类型：
--
作者：
H. Akiyama;Fumi Nakamura;C. Yamada;Kosuke Nakamura;O. Nakajima;H. Kawakami;N. Harikai;S. Furui;K. Kitta;R. Teshima
为了筛查各种作物中未经授权的转基因生物(GMO)，我们建立了一种多重实时聚合酶链式反应高分辨率熔融曲线分析方法，用于同时定性检测几种作物中花椰菜花叶病毒(35SP)的35S启动子序列和诺帕林合成酶终止子(NOST)。为同时检测35SP和NOST，我们选择了合适的引物组，并设计了一套用于检测添加了ColE1的质粒的引物组，以评价聚合酶链式反应(PCR)分析的有效性。此外，以EvaGreen为插层染料，优化了多重聚合酶链式反应条件。类似于NK603玉米的单拷贝未经授权的转基因作物的污染可以在玉米样品中检测到低至0.1%。此外，我们还证明了本方法将适用于鉴定各种农作物和食品中的转基因生物，如授权转基因大豆、授权转基因土豆、被转基因大豆污染的饼干和被未经授权转基因大米污染的大米。我们认为，这种方法是一种简单可靠的检测方法，用于筛选农作物和加工食品中的未经授权的转基因作物。
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.
影响因子：
2.9
作者：
Ririe, KM;Rasmussen, RP;Wittwer, CT
通讯作者：
Wittwer, CT