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Soybean (Glycine max L. Merrill) is one of eight major allergenic foods with endogenous proteins identified as allergens. To better understand the natural variability of five soybean allergens (Gly m 4, Gly m 5, Gly m 6, Gly m Bd 28k, and Gly m Bd 30k), validated enzyme-linked immunosorbent assays (ELISAs) were developed. These ELISAs measured allergens in 604 soybean samples collected from locations in North and South America over five growing seasons (2009-2013/2014) and including 37 conventional varieties. Levels of these five allergens varied 5-19-fold. Multivariate statistical analyses and pairwise comparisons show that environmental factors have a larger effect on allergen levels than genetic factors. Therefore, from year to year, consumers are exposed to highly variable levels of allergens in soy-based foods, bringing into question whether quantitative comparison of endogenous allergen levels of new genetically modified soybean adds meaningful information to their overall safety risk assessment.

Anthocyanidin reductase (ANR; EC 1.3.1.77) catalyzes a key step in the biosynthesis of proanthocyanidins (PAs; also known as condensed tannins), flavonoid metabolites responsible for the brown pigmentation of seeds. Here, two ANR genes (ANR1 and ANR2) from the seed coat of brown soybean (Glycine max (L.) Merr.) have been isolated and their enzymatic function confirmed for the reduction of cyanidin to (-)-epicatechin in vitro. Biochemical and genetic comparisons of soybean lines differing in seed coat color revealed three red-brown lines to exhibit major reductions in the amounts of soluble PAs in the seed coat compared to brown soybean lines. Two spontaneous mutants with red-brown grain color had reduced ANR1 gene expression in the seed coat, and an EMS-mutagenized red-brown mutant had nonsynonymous substitutions that resulted in slightly reduced ANR1 activity in vitro. These results suggest that defects in the ANR1 gene can be associated with red-brown soybean grain color. These results suggest that suppressing ANR1 gene expression or activity may be a rational approach toward engineering seed coat color to enable the visual identification of genetically modified soybean grains.

Various polymerase chain reaction (PCR) methods were developed for the execution of genetically modified organism (GMO) labeling policies, of which an event-specific PCR detection method based on the flanking sequence of exogenous integration is the primary trend in GMO detection due to its high specificity. In this study, the 5' and 3' flanking sequences of the exogenous integration of MON89788 soybean were revealed by thermal asymmetric interlaced PCR. The event-specific PCR primers and TaqMan probe were designed based upon the revealed 5' flanking sequence, and the qualitative and quantitative PCR assays were established employing these designed primers and probes. In qualitative PCR, the limit of detection (LOD) was about 0.01 ng of genomic DNA corresponding to 10 copies of haploid soybean genomic DNA. In the quantitative PCR assay, the LOD was as low as two haploid genome copies, and the limit of quantification was five haploid genome copies. Furthermore, the developed PCR methods were in-house validated by five researchers, and the validated results indicated that the developed event-specific PCR methods can be used for identification and quantification of MON89788 soybean and its derivates.

With the increasing development of genetically modified organism (GMO) detection techniques, the polymerase chain reaction (PCR) technique has been the mainstay for GMO detection. An oligonucleotide microarray is a glass chip to the surface of which an array of oligonucleotides was fixed as spots, each containing numerous copies of a sequence-specific probe that is complementary to a gene of interest. So it is used to detect ten or more targets synchronously. In this research, an event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity using multiplex-PCR together with oligonucleotide microarray. A commercial GM soybean (GTS 40-3-2) and six GM maize events (MON810, MON863, Bt176, Bt11, GA21, and T25) were detected by this method. The results indicate that it is a suitable method for the identification of these GM soybean and maizes.

Three methods of DNA extraction from feed products and four detection methods for the 5'-junction fragment of genetically modified (GM) Roundup Ready soybean (RRS) were compared and evaluated. The DNA extraction methods, including cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and guanidine hydrochloride (Kit), were assessed for their yields and purity of DNA, extraction time, and reagent cost. The DNA yields of CTAB, SDS, and Kit were 52-694, 164-1750 and 23-105 ng/mg sample, and their extraction time was 2.5-3, 2-2.5, and 1.5-2 h with reagent cost about US dollar 0.24, 0.13, and 1.9 per extraction, respectively. The SDS method was generally well suited to all kinds of feed matrices tested. The limits of detection for the four amplification protocols, including loop-mediated isothermal amplification (LAMP), hyperbranched rolling circle amplification (HRCA), conventional polymerase chain reaction (PCR), and real-time PCR, were 48.5, 4.85, 485, and 9 copies of the pTLH10 plasmid, respectively. The ranked results of the four detection methods were based on multiattribute utility theory as follows (from best to worse): HRCA, LAMP, PCR, and real-time PCR. This comparative evaluation was specifically useful for selection of a highly efficient DNA extraction or amplification method for detecting different GM ingredients.

A novel type of quantitative competitive polymerase chain reaction (QC-PCR) system for the detection and quantification of the Roundup Ready soybean (RRS) was developed. This system was designed based on the advantage of a fully validated real-time PCR method used for the quantification of RRS in Japan. A plasmid was constructed as a competitor plasmid for the detection and quantification of genetically modified soy, RRS. The plasmid contained the construct-specific sequence of RRS and the taxon-specific sequence of lectin1 (Le1), and both had 21 bp oligonucleotide insertion in the sequences. The plasmid DNA was used as a reference molecule instead of ground seeds, which enabled us to precisely and stably adjust the copy number of targets. The present study demonstrated that the novel plasmid-based QC-PCR method could be a simple and feasible alternative to the real-time PCR method used for the quantification of genetically modified organism contents.

Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.

Peptide nucleic acid (PNA) microarrays for the detection of Roundup Ready soybeans in food have been prepared. PNA probes are known to be more efficient and selective in binding DNA sequences than the analogous oligonucleotides and are very suitable to be used for diagnostics in food. PNAs of different lengths were carefully designed and synthesized by solid-phase synthesis on an automatic synthesizer adopting the BOC strategy. PNAs were purified by HPLC and characterized by HPLC/MS. The probes were spotted on a functionalized surface to produce a microarray to be hybridized with PCR products. DNA extracted from reference material was amplified using Cy3- and Cy5-labeled primers, and the fluorescent PCR products obtained were hybridized on the microarray. Two protocols were adopted: the hybridization with dsDNA or with ssDNA obtained by digestion with the enzyme lambda exonuclease. The best results were obtained using a 15-mer PNA probe in combination with the ssPCR product derived from enzymatic digestion. The method was applied to the analysis of a sample of certified transgenic soybean flour.