Quantitative Detection System for Maize Sample Containing Combined-Trait Genetically Modified Maize

Abstract

Various countries have established regulations that stipulate the labeling of agricultural commodities, feed, and food products that contain or are made from genetically modified (GM) material or that contain adventitious GM material in amounts that exceed certain threshold levels. While regulations in some countries refer to GM material on a weight per weight (w/w) percentage, the currently applied detection methods do not directly measure the w/w percentage of the GM material. Depending on the particular method and the sample matrix it is applied to, the conversion of analytical results to a w/w percentage is challenging or not possible. The first rapid PCR system for GM maize detection on a single kernel basis has been developed. The equipment for the grinding of individual kernels and a silica membrane-based 96-well DNA extraction kit were both significantly revised and optimized for this particular purpose, respectively. We developed a multiplex real-time PCR method for the rapid quantification of GM DNA sequences in the obtained DNA solutions. In addition, a multiplex qualitative PCR detection method allows for the simultaneous detection of different GM maize traits in each kernel and thereby for identification of individual kernels that contain a combination of two or more GM traits. Especially for grain samples that potentially contain combined-trait GM maize kernels, the proposed methods can deliver informative results in a rapid, precise, and reliable manner.

Development of Multiplex PCR Method for Simultaneous Detection of Four Events of Genetically Modified Maize: DAS-59122-7, MIR604, MON863 and MON88017

“…For the preparation of simulated samples, DNA concentration was measured with PicoGreen dsDNA reagent, which is a sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA and can eliminate the e#ects caused by single-stranded nucleic acids and other impurities. Referring to previous reports῎ 10, ῎ 11 , twenty-one independent multiplex PCR runs were performed for each kind of simulated sample, and the results were analyzed statistically ( Table 2). According to the ISO standard for GMO analysis methods on foodstu#s῎ 12 , the LOD value was defined as the lowest level of analytes with the false negative rate of 5῍ or less in multilaboratory collaborative evaluation.…”

“…For the detection of GM maize in foods and food materials, PCR-based detection methods, which are able to detect even small amounts of transgenes in raw materials and processed foods, have been routinely used. We have also developed simplex and multiplex qualitative PCR methods 2)ῌ7) , simplex real-time PCR methods 8), 9) and an individual kernel-based detection method including a qualitative multiplex real-time PCR 10), ῎ 5 . Some of these methods have been employed as o$cial analytical methods in Japan῎ 6, ῎ 7 .…”

Interlaboratory Validation of an Event-Specific Real Time Polymerase Chain Reaction Detection Method for Genetically Modified DAS59132 Maize

“…The SSIIb-3 system (SSIIb 3῍5῎ and SSIIb 3῍3῎ with SSIIb-Taq) was used for real-time PCR detection of the taxon specific gene encoding the maize starch synthase IIb gene sequence (SSIIb) as described previously 17), 18) .…”

Development of Quantitative Duplex Real-Time PCR Method for Screening Analysis of Genetically Modified Maize

“…We have also developed simplex and multiplex qualitative PCR methods 6)ῌ11) , simplex real-time PCR methods 12), 13) , and an individual kernel-based detection method including a qualitative multiplex real-time PCR 14) . In particular, six real-time PCR methods for five events of GM maize, i.e., Bt11, Bt176, GA21, MON810, and T25, and a Roundup Ready soy, were fully validated by an international interlaboratory collaborative study 12), 15) , and have been not only employed as Japanese o$cial analytical methods῏ 3, ῏ 4 , but also internationally recognized as ISO standard methods for the analysis of GMOs in foodstu#s῏ 5 .…”