Real-Time PCR Array as a Universal Platform for the Detection of Genetically Modified Crops and Its Application in Identifying Unapproved Genetically Modified Crops in Japan

Mano, Junichi; Shigemitsu, Natsuki; Futo, Satoshi; Akiyama, Hiroshi; Teshima, Reiko; Hino, Akihiro; Furui, Satoshi; Kitta, Kazumi

doi:10.1021/jf802551h

Cited by 60 publications

(31 citation statements)

References 27 publications

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“…In addition, we analyzed the GM events in GM maize grains of non-IP maize samples in 2009 using two multiplex qualitative PCR detection methods 4), 6) coupled to microchip electrophoresis and partially real-time PCR array analyses 7) . MON88017 and NK603 were the major single GM events and MON88017 MON810 was the major stacked GM event in the non-IP samples.…”

Section: Discussionmentioning

confidence: 99%

“…To clarify GM events in genomic DNAs from some GM grains that gave ambiguous results in analyses using the two multiplex qualitative PCR detection methods, a real-time PCR array was employed according to our previously reported method with some modifications 7) . The following detection targets were selected for one analysis: Bt11, E176, GA21, M810, M863, NK603, T25, TC1507, DAS59122, M88017, MIR604 and SSIIb.…”

Section: Preparation Of Real-time Pcr Array Reaction Conditions and mentioning

confidence: 99%

“…Next, the genomic DNA extracted from positive kernels of five non-IP maize grain samples was individually analyzed using two multiplex qualitative PCR detection methods 4), 6) both coupled to microchip electrophoresis and a partially real-time PCR array method 7) , to clarify whether these GM events are present as single or stacked events and which event is present in the genomic DNA from each kernel.…”

Section: Gm Event Analysis Using Multiplex Qualitative Pcrmentioning

confidence: 99%

“…The present study was designed to clarify the GM maize content of non-IP maize samples that contain GM maize produced in 2009, to investigate the content of GM maize grains, and to determine how many stacked GM maize grains are contained therein and which GM maize and stacked GM maize events frequently appeared in 2009 by using the multiplex real-time PCR method 2), 3) , two multiplex qualitative PCR detection methods 4), 6) both coupled to the microchip electrophoresis and partially real-time PCR array analysis 7) .…”

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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: Discussionmentioning

confidence: 99%

Section: Preparation Of Real-time Pcr Array Reaction Conditions and mentioning

confidence: 99%

Section: Gm Event Analysis Using Multiplex Qualitative Pcrmentioning

confidence: 99%

mentioning

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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“…Dry seeds of conventional soy were imported directly from the USA and were used as a non-GM material. GM and non-GM materials were ground in a Multi-beads shocker (model MB601, Yasui Kikai, Osaka, Japan), and the purity of each sample was then confirmed by real-time PCR array analysis 12) .…”

Section: Methodsmentioning

confidence: 99%

Development of Direct Real-Time PCR System Applicable to a Wide Range of Foods and Agricultural Products

Mano

Hatano²,

Futo³

et al. 2014

Journal of the Food Hygienics Society of Japan

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To improve the efficiency of DNA analysis of foods and agricultural products, we investigated a direct real-time PCR based on the real-time monitoring of DNA amplification directly from crude cell lysates of analytical samples. We established a direct real-time PCR system comprising sample pretreatment with a specified lysis buffer and real-time PCR using the developed master mix reagent. No PCR inhibition was observed in the analysis of crude cell lysates from 50 types of samples, indicating that the direct real-time PCR system is applicable to a wide range of materials. The specificity of the direct real-time PCR was evaluated by means of a model assay system for single nucleotide discrimination. Even when crude cell lysates coexisted in the reaction mixtures, the primer selectivity was not affected, suggesting that the sequence specificity of the direct real-time PCR was equivalent to that of PCR from purified DNA templates. We evaluated the sensitivity and quantitative performance of the direct real-time PCR using soybean flour samples including various amounts of genetically modified organisms. The results clearly showed that the direct real-time PCR system provides sensitive detection and precise quantitation.

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Genetically Modified Organisms Detection by Analytical and Spectroscopical Methods

Holck

2012

Encyclopedia of Analytical Chemistry

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Genetically modified plants (GMPs) are grown on 10% of the world's fields and the area is continuously increasing. GMPs are subject to extensive regulations due to public health and environmental concerns. Legislation in the European Union requires labeling of food and feed containing authorized GMP above 0.9%. Other countries have different thresholds. European Union has a zero tolerance for unauthorized materials, which have not undergone a risk assessment. Analytical methods play a central role in enforcing legislation. A large number of methods for identification of genetic modification based on DNA detection has been developed. Detection relies often on quantitative real‐time polymerase chain reaction (PCR) methods. The increasing number of GMP entering the market spurs development of both qualitative and quantitative multiplex methods. Qualitative multiplex PCR is usually performed with several primer pairs, detecting one target each. To increase multiplexing and obtain quantitative results, ligation‐mediated PCR has been employed. PCR products are often detected by capillary electrophoresis (CE) or by hybridization to microarrays. In addition, other non‐PCR‐based methods exist. Identification of unknown samples is often a two‐step process. First, commonly used GM (genetically modified) elements are detected, and second, specific GM events are determined based on the results from the first analysis. Immunological detection methods also exist for separating GMP and non‐GMP grain lots at farm and elevator level.

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Real-Time PCR Array as a Universal Platform for the Detection of Genetically Modified Crops and Its Application in Identifying Unapproved Genetically Modified Crops in Japan

Cited by 60 publications

References 27 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

Development of Direct Real-Time PCR System Applicable to a Wide Range of Foods and Agricultural Products

Genetically Modified Organisms Detection by Analytical and Spectroscopical Methods

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