Development of an agricultural biotechnology crop product: testing from discovery to commercialization.

"Genetically modified" (GM) or "biotech" crops have been the most rapidly adopted agricultural technology in recent years. The development of a GM crop encompasses trait identification, gene isolation, plant cell transformation, plant regeneration, efficacy evaluation, commercial event identification, safety evaluation, and finally commercial authorization. This is a lengthy, complex, and resource-intensive process. Crops produced through biotechnology are the most highly studied food or food component consumed. Before commercialization, these products are shown to be as safe as conventional crops with respect to feed, food, and the environment. This paper describes this global process and the various analytical tests that must accompany the product during the course of development, throughout its market life, and beyond.

Practicable group testing method to evaluate weight/weight GMO content in maize grains.

Because of the increasing use of maize hybrids with genetically modified (GM) stacked events, the established and commonly used bulk sample methods for PCR quantification of GM maize in non-GM maize are prone to overestimate the GM organism (GMO) content, compared to the actual weight/weight percentage of GM maize in the grain sample. As an alternative method, we designed and assessed a group testing strategy in which the GMO content is statistically evaluated based on qualitative analyses of multiple small pools, consisting of 20 maize kernels each. This approach enables the GMO content evaluation on a weight/weight basis, irrespective of the presence of stacked-event kernels. To enhance the method's user-friendliness in routine application, we devised an easy-to-use PCR-based qualitative analytical method comprising a sample preparation step in which 20 maize kernels are ground in a lysis buffer and a subsequent PCR assay in which the lysate is directly used as a DNA template. This method was validated in a multilaboratory collaborative trial.

Interlaboratory validation of an event-specific real time polymerase chain reaction detection method for genetically modified DAS59132 maize.

A real-time polymerase chain reaction (PCR) method specific for genetically modified (GM) maize event DAS59132 (E32) was adapted for qualitative detection of low level presence of E32. The method was validated by a collaborative trial with eight participating Japanese laboratories. Sensitivity was assessed with three different samples of corn flour fortified to 0%, 0.05% and 0.1% (w/w) E32 respectively. In addition, a 0.01% E32 DNA solution was used. The detection limit with DNA solution was estimated to be approximately 0.01%. In conclusion, the results of the study confirmed this real-time PCR method as a reliable tool for qualitative detection of E32 maize.

Development and evaluation of event-specific qualitative PCR methods for genetically modified Bt10 maize.

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.

Quantitative detection system for maize sample containing combined-trait genetically modified maize.

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.

Polymerase chain reaction technology as analytical tool in agricultural biotechnology.

The agricultural biotechnology industry applies polymerase chain reaction (PCR) technology at numerous points in product development. Commodity and food companies as well as third-party diagnostic testing companies also rely on PCR technology for a number of purposes. The primary use of the technology is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of PCR analysis and its application to the testing of grains. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effect they may have on the accuracy of the PCR analytical results.