中文摘要：食品和饲料中存在的转基因生物主要通过使用目标启动子和终止子来进行检测。由于一些基因经常用于转基因工程中，因而它们构成了优良的筛选元素且用途也再增多。本文提出了一种新的基于实时PCR和焦磷酸测序的Cry1Ab和Cry1Ac基因检测方法。作者在国际指南的参考下检测了实时PCR方法的特异性、敏感性和鲁棒性，检测发现该方法可满足预期的性能标准。相对采用双染料探针的方法来说，新型Cry1Ab/Ac方法更能为大多数转基因植物提供一个积极信号；相对SYBRGreen方法来说，该方法的分析结果更加明确。焦磷酸测序方法也被开发来获得更多的信息，这种合成测序方法可以确定PCR引物间的序列。
外文摘要：The presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.
外文关键词：GMO detection; genes; screening; pyrosequencing; real-time PCR
作者：Debode, Frederic; Janssen, Eric; Bragard, Claude; 等
作者单位：Walloon Agr Res Ctr CRA W
期刊名称：FOOD ADDITIVES AND CONTAMINANTS PART A-CHEMISTRY ANALYSIS CONTROL EXPOSURE & RISK ASSESSMENT
期刊影响因子：0.0
出版年份：2017
出版刊次：8
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