Laboratory-performance Study of Quantitative PCR Methods to Analyze an Approved Genetically Modified Maize (Mon810 Line)

Watanabe, Takahiro; Kasama, Kikuko; Kikuchi, Hiroyuki; Suzuki, Tatsuya; Tokishita, Shoko; Sakata, Kozue; Matsuki, A; Hino, Akihiro; Akiyama, Hiroshi; Maitani, Tamio

doi:10.3358/shokueishi.47.15

Cited by 3 publications

(4 citation statements)

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“…34) However, it also has been reported that the value of the MON810 maize content measured using a LightCycler Ò real-time PCR system was significantly high in a laboratoryperformance study. 33) In the present study, the reported RRS quantification conditions were first applied to GM corn quantification, and comparable studies between pDNA and gDNA for quantitative analysis of GM maize were performed. As shown in Table 1, it is clear that the slopes for gDNA and pDNA are significantly different for all the SSIIb, P35S, and MON810.…”

Section: Resultsmentioning

confidence: 99%

“…When the quantification method of the ABI PRISM 7700 system was applied to the LightCycler Ò real-time PCR system, it was reported that the value of the GM maize contents measured by the LightCycler Ò real-time PCR system appeared to be significantly different from the expected theoretical value on a weight-to-weight basis. 33) In addition, a recent study suggested that DNA extraction methods affect the quantified value of the GM maize contents using real-time PCR equipment because the integrity and purity of the extracted genomic DNA differs depending on the DNA extraction method. 34) We have also developed a system for Roundup Ready Ò Soybean (RRS) quantification using a combination of the LightCycler Ò real-time PCR system and pDNA as the reference standard.…”

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confidence: 99%

“…[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] Hence many real-time PCR systems based on fluorescent detection, such as TaqMan Ò chemistry, have been developed to identify and quantify GM maize, GM soybeans, and GM varieties of other agricultural commodities. [22][23][24][25][26][27][28][29][30][31][32][33] Real-time PCR systems using TaqMan Ò chemistry are based on the use of a fluorescent TaqMan Ò probe that monitors the formation of the PCR product during each cycle of the reaction. In addition, most commonly, GMO quantification by real-time PCR methods is calculated from the ratio of the target transgenic specific DNA sequence copy number vs. the DNA sequence copy number of the respective target plant species (taxon-specific sequence).…”

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Rapid Quantification Methods for Genetically Modified Maize Contents Using Genomic DNAs Pretreated by Sonication and Restriction Endonuclease Digestion for a Capillary-Type Real-Time PCR System with a Plasmid Reference Standard

Toyota

Akiyama²,

Sugimura³

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Rapid Quantification Methods for Genetically Modified Maize Contents Using Genomic DNAs Pretreated by Sonication and Restriction Endonuclease Digestion for a Capillary-Type Real-Time PCR System with a Plasmid Reference Standard

Toyota

Akiyama²,

Sugimura³

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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“…Extraction and Purification of Genomic DNA. Genomic DNAs were extracted from the ground materials and Bt10 powder-mixture samples using a silica-gel membrane-type kit (DNeasy Plant Mini; QIAGEN, Hilden, Germany) according to the procedure described in our previous study with some modification (19). The DNA concentration was determined by measuring the UV absorption at 260 nm using an ND-1000 spectrophotometer (NanoDrop Technologies Inc., Rockland, DE).…”

Section: Methodsmentioning

confidence: 99%

Development and Evaluation of Event-Specific Qualitative PCR Methods for Genetically Modified Bt10 Maize

Watanabe

Tokishita

Spiegelhalter

et al. 2007

J. Agric. Food Chem.

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In 2005 it was reported that the genetically modified (GM) maize strain or "event" called Bt10 had been distributed inadvertently in the United States over the previous 4 years. In order to ensure that grain for food and feed production did not contain trace amounts of Bt10 maize and complied with the applicable regulation, highly sensitive and specific detection of Bt10 maize was required. Accordingly, we developed a novel qualitative PCR system for specific detection of Bt10 maize. Moreover, we amply evaluated the performance characteristics of two PCR systems, our own and the one provided by the developer of Bt10, Syngenta Co. Ltd. It was confirmed that both of the qualitative PCR systems can specifically detect Bt10 maize, and the results of a single-laboratory examination suggested that the limit of detection was approximately less than 0.05% for both methods. To evaluate the reproducibility of the methods, we organized an interlaboratory study with the participation of 6 laboratories and analysis of 240 blind test samples. In this paper, we report, for the first time, the statistical analysis of the qualitative PCR data obtained from the interlaboratory study. The results of this analysis also revealed that there was no significant difference in the sensitivity between the two aforementioned methods and that the limit of detection of both the methods was less than 0.05%. Thus, we conclude that both of the methods are equally suitable for correct identification and sensitive detection of the unapproved GM maize Bt10 event in test samples.

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Development and Evaluation of Qualitative Detection Methods for Unapproved Genetically Modified Rice (LLRice)

Watanabe¹,

Shiramasa²,

Furui³

et al. 2007

J. Food Hyg. Soc. Jpn

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We developed a specific method to extract DNA from rice grain samples and modified the qualitative real-time PCR method provided by Bayer Co., Ltd. for reliable detection of the genetically modified (GM) rice variety, LLRice601, which has not undergone safety assessment for regulatory approval in Japan. Moreover, we conducted a data analysis to confirm the results obtained with real-time PCR. The yields of DNA extracted from powdered samples of rice grains were almost equal among 5 di#erent varieties of rice, and there was no significant di#erence in the yield over three days. Reliable results were obtained using 50 ng of the extracted DNA as the template for real-time PCR. To examine the adequacy of the methods, we organized an interlaboratory study with the participation of 2 laboratories, in which 80 test samples were analyzed in a blinded manner. The statistical analysis revealed no significant di#erence in the Ct value for the endogenous gene of the DNA samples and for the targeted DNA sequence of 0.1ΐ samples. The limit of detection of the method was approximately 0.1ΐ. Analysis of the fluorescence intensity of the PCR-amplified product of the construct-specific DNA sequence suggested that it may be reasonable to judge a sample as positive when a Ct value of less than 40 is obtained.

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Laboratory-performance Study of Quantitative PCR Methods to Analyze an Approved Genetically Modified Maize (Mon810 Line)

Cited by 3 publications

References 5 publications

Rapid Quantification Methods for Genetically Modified Maize Contents Using Genomic DNAs Pretreated by Sonication and Restriction Endonuclease Digestion for a Capillary-Type Real-Time PCR System with a Plasmid Reference Standard

Rapid Quantification Methods for Genetically Modified Maize Contents Using Genomic DNAs Pretreated by Sonication and Restriction Endonuclease Digestion for a Capillary-Type Real-Time PCR System with a Plasmid Reference Standard

Development and Evaluation of Event-Specific Qualitative PCR Methods for Genetically Modified Bt10 Maize

Development and Evaluation of Qualitative Detection Methods for Unapproved Genetically Modified Rice (LLRice)

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