Development of a Multiplex Polymerase Chain Reaction Method for Simultaneous Detection of Eight Events of Genetically Modified Maize

Onishi, Mari; Matsuoka, Takeshi; Kodama, Takashi; Kashiwaba, Koichi; Futo, Satoshi; Akiyama, Hiroshi; Maitani, Tamio; Furui, Satoshi; Oguchi, Taichi; Hino, Akihiro

doi:10.1021/jf0515476

Cited by 75 publications

(44 citation statements)

References 24 publications

(36 reference statements)

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“…To identify which GM event grains are contained in genomic DNA extracted from individual kernels, two multiple qualitative PCR detections were performed according to our previously reported methods 4) . The first method was performed for the detection of MON810, NK603, T25, GA21, TC1507, Event176, Bt11, and MON863…”

Section: Two Multiple Qualitative Pcr Methodsmentioning

confidence: 99%

“…However, there has been little information on the determination of stacked GM maize in non-IP maize samples, because no method is available, except for detection in individual kernels. We previously investigated GM maize content on a kernel basis and determined the varieties of the GM kernels in non-IP maize samples imported from the USA in 2005 5) using an individual kernel analysis system including a multiplex real-time PCR method 2), 3) , and coupled it to a multiplex qualitative PCR method 4) followed by analysis using multi-channel capillary gel electrophoresis 5) .…”

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confidence: 99%

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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

Akiyama¹,

Minegishi

Makiyama³

et al. 2012

Journal of the Food Hygienics Society of Japan

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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...

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Section: Two Multiple Qualitative Pcr Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Akiyama¹,

Minegishi

Makiyama³

et al. 2012

Journal of the Food Hygienics Society of Japan

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“…be confirmed in detail using some reference methods (PCR 40,41 or protein immunoassays) and much larger number of samples in order to obtain statistically significant data, and also some information about possible quantification aspects of such measurements. So far, the main obstacle in such studies is difficulty in obtaining model samples of different GMO variants, even for the most commonly cultivated maize or soybean.…”

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confidence: 98%

Modification of Resolution in Capillary Electrophoresis for Protein Profiling in Identification of Genetic Modification in Foods

et al. 2011

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Abstract. The capillary electrophoresis with UV detection was employed for protein profiling in extracts from maize and soybeans. Modifications of back-ground electrolyte and coating the capillary wall with polybrene was employed in order to decrease the protein adsorption on the capillary walls. The obtained protein profiles were compared for transgenic and non-transgenic variants, showing in some cases significant changes that might be employed for identification of genetic modifications of plants or foods of plant origin. (doi: 10.5562/cca1777)

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“…Beberapa metode deteksi telah dikembangkan di dunia, antara lain oleh Ahmed (2002), Lee et al (2004), dan Onishi et al (2005). Di Indonesia, beberapa institusi telah mengembangkan teknik deteksi PCR secara kualitatif (Bahagiawati dan Sutrisno, 2007).…”

Section: Pendahuluanunclassified

Teknik PCR Kualitatif untuk Deteksi Produk Rekayasa Genetika Jagung Event BT11 dan GA21

Bahagiawati¹,

Reflinur²,

Santoso³

2015

J. AgroBiogen

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In some countries, including Indonesia, labelling of GMO products is mandatory for giving consumers the right to choose between GMOs and conventional products. Therefore, development of methodology that can detect a specific genetically modified (GM) crops and to verify the absence or presence of GM material in a product including raw materials (e.g. grains) and/or their derivatives is needed. The objectives of this study were to find the most efficient screening methods to detect whether or not a product is GM material and to develop a specific detection method to identify GM product BT11 and GA21. In addition, present study was also aimed to obtain a duplex detection method for both GM products. Two GM-maize, including the BT11 and GA21 lines of maize (Zea mays L.), and one plant, namely NK11 as the nontransgenic control, were used as plant genetic materials in the event-specific detection of maize. The target gene from each sample was amplified in different reaction (simplex) using both the event specific primer and the endogenous maize reference, Zein, as internal control. Furthermore, in duplex PCR, two targets were simultaneously amplified in the same reaction. The results showed that detection method of the GM product obtained from present study enabled us to screen the GM products and specifically the event of BT11 and GA21 using simplex and duplex methods. The duplex method is more efficient because it can detect two GM crops in one time compared to simplex method that only can detect GM crop one by one.Keywords: GMO detection, GMO screening, maize, BT11, GA21. ABSTRAKPelabelan produk hasil rekayasa genetika (PRG) di beberapa negara, termasuk Indonesia, merupakan mandat yang memberikan kesempatan kepada konsumen untuk mendapatkan informasi produk yang akan dibeli atau digunakan. Untuk itu, diperlukan perangkat teknologi yang dapat mendeteksi keberadaan produk PRG tersebut, baik dalam bentuk bahan dasar (bijibijian) maupun produk turunannya. Penelitian ini bertujuan membandingkan dan mendapatkan teknik yang akurat dan efisien untuk deteksi tanaman jagung BT11 dan GA21 berdasarkan teknik PCR, baik secara tunggal (simpleks) maupun ganda (dupleks). Jagung produk PRG, yaitu BT11 dan GA21, dan non-PRG, yaitu Nk11, digunakan sebagai sampel dalam pengembangan metode deteksi PRG pada kedua event tersebut. Setiap sampel diamplifikasi secara simpleks menggunakan primer spesifik untuk mendeteksi keberadaan gen target pada event PRG dan sepasang primer spesifik sebagai internal kontrol untuk tanaman jagung. Selanjutnya, pada metode dupleks, sampel DNA kedua event diamplifikasi dengan primer-primer tersebut secara bersamaan dalam suatu reaksi PCR. Hasil penelitian menunjukkan bahwa teknik deteksi yang didapatkan dari penelitian ini dapat digunakan untuk skrining PRG dan identifikasi spesifik BT11 dan GA21, baik secara simpleks maupun dupleks. Kemampuan metode dupleks untuk mendeteksi PRG akan sangat bermanfaat dalam kegiatan skrining PRG dalam jumlah sampel yang besar karena metode ini lebih efisien dibanding...

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Development of a Multiplex Polymerase Chain Reaction Method for Simultaneous Detection of Eight Events of Genetically Modified Maize

Cited by 75 publications

References 24 publications

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

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

Modification of Resolution in Capillary Electrophoresis for Protein Profiling in Identification of Genetic Modification in Foods

Teknik PCR Kualitatif untuk Deteksi Produk Rekayasa Genetika Jagung Event BT11 dan GA21

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