粒単位検出システムによる2009年度産遺伝子組換えトウモロコシ試料の遺伝子組換えトウモロコシ品種の定量と同定

Abstract

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...

A novel trait-specific real-time PCR method enables quantification of genetically modified (GM) maize content in ground grain samples containing stacked GM maize

“…The National Agricultural Statistics Service (NASS) reported that the percentage of stacked GM maize events has consecutively increased from 1 % in 2000 to 71 % in 2013 in the USA [43]. Indeed, our previous studies showed that the percentage of stacked GM maize events in non-IP samples imported to Japan from the USA increased from 12 % in 2005 [24] to 35 % in 2009 [25]. Judging from these data, almost all GM maize is composed of stacked GM maize events, indicating that no single GM maize events will be distributed commercially in the future.…”

“…The discrepancy between ideal and experimental values has been previously reported [2,31] and may be due to differences in PCR efficiencies resulting from the amount of non-targeted sequences in plasmid and genomic DNA [2] or due to differences in the efficiency of DNA extraction between reference and trait genes [31]. GM maize imported to Japan contains not only single GM maize events having a single copy of the trait gene per genome, but also stacked GM maize events having multiple copies of the trait gene per genome as previously reported [25]. The corrected C f values for each trait gene were required to calculate GMO contents in maize samples containing GM maize events having multiple copies of the trait gene per genome, because GMO content is overestimated using the C f value calculated from each GM maize event having a single copy of the trait gene per genome.…”

“…where C f(trait) is each C f value for cp4epsps or pat in a GM maize event containing cp4epsps or pat and x is the rate of a GM maize event in all main GM maize events containing cp4epsps or pat, which was calculated using the data of GMO content in the non-IP maize sample imported from the USA in 2009 [25] ( Table 3). The corrected C f was calculated by summing the product of C f(trait) and × (C f(trait) × x) for each GM maize event.…”

“…We previously determined the GM maize content on a kernel basis and the events of GM maize kernels in non-identity-preserved (IP) maize samples imported from the USA in 2005 and 2009 using an individual kernel detection system [24,25]. The main GM maize events detected in the non-IP maize samples in 2009 were MON88017, MON810 × MON88017, NK603, MON810, TC1507 × DAS59122, MON810 × NK603, TC1507, DAS59122 and MON863.…”

Capillary Electrophoresis Based on Nucleic Acid Detection as Used in Food Analysis

“…Thus, rapid and sensitive methods of detecting GMOs are required to allow timely and accurate labeling (Valdés and others ; Milavec and others ; Fraiture and others ). PCR‐CE‐based procedures have been assessed as competitive methods of detecting GMOs because they have high sensitivity, high throughput, and high speed (Burrell and others ; Akiyama and others ; Stojkovic and others ). MSPE‐PCR‐CE was used to successfully detect GM soybeans, which demonstrated its potential for GMO analysis (Jiang and others ; Deng and others ).…”