National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Anal Chem. 2010 Dec 1;82(23):9909-16. doi: 10.1021/ac102434q. Epub 2010 Nov 4.
The herbicide-tolerant genetically modified Roundup Ready canola (Brassica napus) line RT73 has been approved worldwide for use in animal feed and human food. However, RT73 Brassica rapa lines derived from interspecific crosses with RT73 B. napus have not been approved in Japan. Here, we report on a novel system using individual kernel analyses for the qualitative detection of RT73 B. rapa in canola grain samples. We developed a duplex real-time polymerase chain reaction (PCR) method to discriminate B. napus and B. rapa DNA using scatter plots of the end-point analyses; this method was able to discriminate a group comprising B. rapa and Brassica juncea from a group comprising B. napus, Brassica carinata, and Brassica oleracea. We also developed a duplex real-time PCR method for the simultaneous detection of an RT73-specific sequence and an endogenous FatA gene. Additionally, a DNA-extraction method using 96-well silica-membrane plates was developed and optimized for use with individual canola kernels. Our detection system could identify RT73 B. rapa kernels in canola grain samples enabling the accurate and reliable monitoring of RT73 B. rapa contamination in canola, thus playing a role in its governmental regulation in Japan.
抗草甘膦基因改造的耐除草剂油菜（甘蓝型油菜）RT73 已在全球范围内获准用于动物饲料和人类食品。然而，与 RT73 油菜进行种间杂交得到的 RT73 甘蓝型油菜品系尚未在日本获得批准。在这里，我们报告了一种使用单个籽粒分析进行定性检测油菜籽粒样品中 RT73 甘蓝型油菜的新系统。我们开发了一种双重实时聚合酶链反应（PCR）方法，使用终点分析的散点图来区分油菜和甘蓝型油菜 DNA；该方法能够将包括甘蓝型油菜和芥菜在内的一组与包括油菜、甘蓝黑芥和甘蓝型油菜在内的一组区分开来。我们还开发了一种用于同时检测 RT73 特异性序列和内源性 FatA 基因的双重实时 PCR 方法。此外，还开发了一种使用 96 孔硅膜板的 DNA 提取方法，并对其进行了优化，以适用于单个油菜籽粒。我们的检测系统能够识别油菜籽粒样品中的 RT73 甘蓝型油菜籽粒，从而能够准确可靠地监测油菜中 RT73 甘蓝型油菜的污染情况，因此在日本的政府监管中发挥了作用。
