We investigated the GM maize grain content of non-identity preserved (non-IP) maize samples produced in 2009 in the USA using our individual kernel detection system, involving two multiplex qualitative PCR methods coupled to microchip electrophoresis and partially real-time PCR array analysis, to clarify how many GM event maize grains were present in the samples and which GM events frequently appeared in 2009. The average percentage and standard deviation of GM maize grains on a kernel basis in five non-IP sample lots were 81.9 2.8 , the average percentage of single GM event grains was 46.9 , and the average percentage of stacked GM event grains was 35.0 . MON88017 grains and NK603 grains were the most frequently observed as single GM event grains. The most frequent stacked GM event grains were MON88017 MON810 grains. This study shows that our method can provide information about GM maize events present in imported maize samples on a kernel basis. Key words genetically modified maize; event; multiplex qualitative PCR; microchip electrophoresis IntroductionGenetically modified (GM) crops are currently cultivated widely as sources of food and feed in many countries 1) . GM crops generally have been assessed and authorized for food use by administrative authorities. In some countries, the labeling of grains, feed and foodstuffs is mandatory if the GM crop content exceeds a certain level of the approved GM varieties. For instance, the European Union, Japan and Korea have set threshold values of 0.9 , 5 , and 3 , respectively, of GM organism material in a non-GM background as the basis for labeling * 1-* 7 .In Japan, non-GM crops are segregated as non-GM material and imported from the United States using an identity preserved (IP) handling system that requires documentary certification from US farms to Japanese processing traders. Recently, the production of stacked GM maize grains, in which two or more characteristic # These authors contributed equally to this study. . Although the levels of adventitious commingling of GM maize in non-GM maize according to the labeling system refer to GM maize as a weight per weight (w/w) percentage, conventional applicable detection methods, such as quantitative real-time PCR, do not directly measure the w/w percentage of GM maize, but rather provide relative copy numbers between a specific DNA sequence and a taxon-specific DNA sequence, and these values are converted into a w/w percentage using appropriate reference materials. The GM maize content in a maize sample containing stacked GM maize grains, as determined by current quantitative real-time PCR methods, is likely to be overestimated compared to the actual w/w percentage of GM maize in the sample because the relative copy numbers are calculated on a haploid basis. To solve this problem, we have developed an individual kernel detection system that consists of grinding individual maize kernels, DNA extraction from each individual ground maize kernel, multiplex real-time PCR using the extracted DNA samples from individual groun...
During the fall of 2009, a trace of unauthorized genetically modified (GM) flax (Linum usitatissimum L.) line, CDC Triffid, which is resistant to sulfonylurea herbicides, was detected in many countries including Japan. A method to reliably identify the CDC Triffid line was urgently required. We developed a novel construct-specific real-time polymerase chain reaction (PCR) method to identify the mutant acetolactate synthase gene in the CDC Triffid line. We confirmed that the method can detect 0.001% GM flax in DNA mixing solution. The study shows that the developed method is specific, sensitive and reliable way to monitor a trace of CDC Triffid.
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.
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