In this study, we developed a novel multiplex polymerase chain reaction (PCR) method for simultaneous detection of up to eight events of genetically modified (GM) maize within a single reaction. The eight detection primer pairs designed to be construct specific for eight respective GM events (i.e., Bt11, Event176, GA21, MON810, MON863, NK603, T25, and TC1507) and a primer pair for an endogenous reference gene, ssIIb, were included in the nonaplex(9plex) PCR system, and its amplified products could be distinguished by agarose gel and capillary electrophoreses based on their different lengths. The optimal condition enabled us to reliably amplify two fragments corresponding to a construct specific sequence and a taxon specific ssIIb in each of the eight events of GM maize and all of nine fragments in a simulated GM mixture containing as little as 0.25% (w/w) each of eight events of GM maize. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM maize.
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.
Genetically modified (GM) sugar beets have been bred for use as food and animal feed. To evaluate the applicability of GMO analyses to beet sugar products, we investigated residual DNA in eight sorts of in-process beet sugar samples and commercial beet sugar products. Polymerase chain reaction (PCR) analyses with taxon-specific primers indicated that sugar beet DNA was degraded at an early stage of sugar processing, and no PCR amplification was detected from the investigated sugar products because of low DNA recovery and/or PCR inhibition.
A novel real-time polymerase chain reaction (PCR)-based quantitative screening method was developed for three genetically modified soybeans: RRS, A2704-12, and MON89788. The 35S promoter (P35S) of cauliflower mosaic virus is introduced into RRS and A2704-12 but not MON89788. We then designed a screening method comprised of the combination of the quantification of P35S and the event-specific quantification of MON89788. The conversion factor (Cf) required to convert the amount of a genetically modified organism (GMO) from a copy number ratio to a weight ratio was determined experimentally. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined RSDr values for the method were less than 25% for both targets. We consider that the developed method would be suitable for the simple detection and approximate quantification of GMO.Key words screening; quantification; genetically modified; real-time polymerase chain reaction; soybeanIn the past few decades, a huge variety of genetically modified (GM) crops have been developed and cultivated worldwide.1) Several countries legislate the labeling systems of authorized GM crops, or their derived foods and feeds. To enforce the labeling system, it is indispensable to define a practical threshold level of GM content and to develop validated quantitative methods for the estimation of unintentional commingling of GM organisms (GMO). GM crops are generally classified in terms of event which is identified by what transgene was inserted, and where on the chromosome it was inserted. There are many GM events approved in Japan.2)The quantitative methods of a glyphosate-tolerant soybean (GTS) Roundup ready soy (RRS) and five GM maize events, i.e., Bt11, Bt176, GA21, MON810, and T25, using real-time polymerase chain reaction (PCR) were developed and validated by an interlaboratory collaborative study, 3) and adopted as Japanese standard methods. 4)For DNA-based detection of GM crops, screening tests are highly cost-and time-effective methods. The Ministry of Health, Labour and Welfare (MHLW) of Japan announced a combinational quantification of P35S and GA21 maize as a quantitative screening method for GM maize and it has been officially adopted.4) Among approved GM soybean events, RRS has been cultivated since in 2001, and the commercial cultivation of a glufosinate-tolerant soybean, A2704-12 and the second generation of GTS, MON89788, have recently started. To analyze these novel GM soybean events, we have developed individual event-specific quantitative methods. 5,6) A practical quantitative screening method for GM soybeans is needed because many food products derived from soybeans are listed for labeling and then frequently analyzed in Japan. 7)In this study, we first developed a new quantitative screening method for RRS, A2704-12 and MON89788. The developed method was evaluated with an interlaboratory collaborative study. MATeRIALS ANd MeTHOdSPlant Materials MON89788 and RRS seeds were kindly provid...
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