Evaluation of modified PCR quantitation of genetically modified maize and soybean using reference molecules: interlaboratory study.

Real-time polymerase chain reaction (PCR)-based quantitative methods were previously developed and validated for genetically modified (GM) maize or soy. In this study, the quantification step of the validated methods was modified, and an interlaboratory study was conducted. The modification included the introduction of the PCR system SSIIb 3 instead of SSIIb 1 for the detection of the taxon-specific sequence of maize, as well as the adoption of colE1 as a carrier included in a reference plasmid solution as a replacement for salmon testis. The interlaboratory study was conducted with the ABI PRISM 7700 and consisted of 2 separate stages: (1) the measurement of conversion factor (Cf) value, which is the ratio of recombinant DNA (r-DNA) sequence to taxon-specific sequence in each genuine GM seed, and (2) the quantification of blind samples. Additionally, Cf values of other instruments, such as the ABI PRISM 7900 and the ABI PRISM 7000, were measured in a multilaboratory trial. After outlier laboratories were eliminated, the repeatability and reproducibility for 5.0% samples were <15.8 and 20.6%, respectively. The quantitation limits of these methods were 0.5% for Bt11, T25, and MON810, and 0.1% for GA21, Event176, and RR soy. The quantitation limits, trueness, and precision of the current modified methods were equivalent to those of the previous methods. Therefore, it was concluded that the modified methods would be a suitable replacement for the validated methods.

Development of taxon-specific sequences of common wheat for the detection of genetically modified wheat.

Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.

Applicability of the quantification of genetically modified organisms to foods processed from maize and soy.

The applicability of quantifying genetically modified (GM) maize and soy to processed foods was investigated using heat treatment processing models. The detection methods were based on real-time quantitative polymerase chain reaction (PCR) analysis. Ground seeds of insect resistant GM maize (MON810) and glyphosate tolerant Roundup Ready (RR) soy were dissolved in water and were heat treated by autoclaving for various time intervals. The calculated copy numbers of the recombinant and taxon specific deoxyribonucleic acid (DNA) sequences in the extracted DNA solution were found to decrease with time. This decrease was influenced by the PCR-amplified size. The conversion factor (Cf), which is the ratio of the recombinant DNA sequence to the taxon specific DNA sequence and is used as a constant number for calculating GM% at each event, tended to be stable when the sizes of PCR products of two DNA sequences were nearly equal. The results suggested that the size of the PCR product plays a key role in the quantification of GM organisms in processed foods. It is believed that the Cf of the endosperm (3n) is influenced by whether the GM originated from a paternal or maternal source. The embryos and endosperms were separated from the F1 generation seeds of five GM maize events, and their Cf values were measured. Both paternal and maternal GM events were identified. In these, the endosperm Cf was lower than that of the embryo, and the embryo Cf was lower than that of the endosperm. These results demonstrate the difficulties encountered in the determination of GM% in maize grains (F2 generation) and in processed foods from maize and soy.

Comparative studies of the quantification of genetically modified organisms in foods processed from maize and soy using trial producing.

Seven types of processed foods, namely, cornstarch, cornmeal, corn puffs, corn chips, tofu, soy milk, and boiled beans, were trial produced from 1 and 5% (w/w) genetically modified (GM) mixed raw materials. In this report, insect resistant maize (MON810) and herbicide tolerant soy (Roundup Ready soy, 40-3-2) were used as representatives of GM maize and soy, respectively. Deoxyribonucleic acid (DNA) was extracted from the raw materials and the trial-produced processed food using two types of methods, i.e., the silica membrane method and the anion exchange method. The GM% values of these samples were quantified, and the significant differences between the raw materials and the trial-produced processed foods were statistically confirmed. There were some significant differences in the comparisons of all processed foods. However, our quantitative methods could be applied as a screening assay to tofu and soy milk because the differences in GM% between the trial-produced processed foods and their raw materials were lower than 13 and 23%, respectively. In addition, when quantitating with two primer pairs (SSIIb 3, 114 bp; SSIIb 4, 83 bp for maize and Le1n02, 118 bp; Le1n03, 89 bp for soy), which were targeted within the same taxon specific DNA sequence with different amplicon sizes, the ratios of the copy numbers of the two primer pairs (SSIIb 3/4 and Le1n02/03) decreased with time in a heat-treated processing model using an autoclave. In this report, we suggest that the degradation level of DNA in processed foods could be estimated from these ratios, and the probability of GM quantification could be experimentally predicted from the results of the trial producing.

[Detection of genetically modified organisms obtained from food samples ].

Genetially modified organisms (GMOs) were explored in food samples obtained from November 2000 to March 2003 in the Tokyo area by using PCR and real-time PCR techniques. The existence of Roundup Ready Soybean (RRS) was surveyed in processed foods derived from soybeans, such as tofu, boiled soybean, kinako, nama-age, abura-age, natto, miso, soymilk and yuba. RRS was detected in 3 of 37 tofu, 2 of 3 nama-age, 2 of 3 yuba and 3 of 3 abura-age samples. The CBH351 in 70 processed corn foods, NewLeaf Plus and NewLeaf Y in 50 processed potato foods, and 55-1 papaya in 16 papayas were surveyed. These GMOs were not detected among the samples. Qualitative and quantitative analyses of RRS and genetically modified (GM) corn were performed in soybean, corn and semi-processed corn products such as corn meal, corn flour and corn grits. RRS was detected in 42 of 178 soybean samples, and the amount of RRS in RRS-positive samples was determined. The content was in the range of 0.1-1.4% in identity-preserved soybeans (non-GMO), and 49.8-78.8% in non-segregated soybeans. On the other hand, GM corns were detected in 8 of 26 samples. The amount of GM corn in GM corn-positive samples was in the range of 0.1-2.0%.

New qualitative detection methods of genetically modified potatoes.

In Japan, 8 lines of genetically modified (GM) potato (2 lines of NewLeaf potato; NL, 3 lines of NewLeaf Plus potato; NLP, and 3 lines of NewLeaf Y potato; NLY) have already been authorized as safe for use in foods and feeds. We have developed polymerase chain reaction (PCR) methods for the qualitative detection of the GM potatoes for the screening and the identification of NL, NLP and NLY. The gene encoding uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) was used as a taxon specific gene. We designed the primer pair to detect the cryIIIA genes as a screening method for GM potatoes because the gene should be inserted in all 8 lines of the GM potatoes. For identification of NL, NLP and NLY, we further designed three specific primer pairs for the different recombinant DNAs (r-DNA) specifically introduced into NL, NLP, or NLY. In addition, to identify the 3 lines of NLY that have been introduced with the same r-DNA, the three line-specific primer pairs for the border sequence between the r-DNA and genomic DNA of NLY 3 lines were designed. Six lines of GM potato used as the test material were specifically identified using the each primer pair under the same PCR condition. The detection limits of all the GM potatoes should be approximately 0.1%. Furthermore, the specificity and reproducibility of the methods were confirmed in a six-laboratory collaborative study.

Novel reference molecules for quantitation of genetically modified maize and soybean.

New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye-labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0% level, the bias (mean-true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.

Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules.

Novel analytical methods based on real-time quantitative polymerase chain reactions by use of new reference molecules were validated in interlaboratory studies for the quantitation of genetically modified (GM) maize and soy. More than 13 laboratories from Japan, Korea, and the United States participated in the studies. The interlaboratory studies included 2 separate stages: (1) measurement tests of coefficient values, the ratio of recombinant DNA (r-DNA) sequence, and endogenous DNA sequence in the seeds of GM maize and GM soy; and (2) blind tests with 6 pairs of maize and soy samples, including different levels of GM maize or GM soy. Test results showed that the methods are applicable to the specific quantitation of the 5 lines of GM maize and one line of GM soy. After statistical treatment to remove outliers, the repeatability and reproducibility of these methods at a level of 5.0% were <13.7 and 15.9%, respectively. The quantitation limits of the methods were 0.50% for Bt11, T25, and MON810, and 0.10% for GA21, Event176, and Roundup Ready soy. The results of blind tests showed that the numerical information obtained from these methods will contribute to practical analyses for labeling systems of GM crops.

Detection of recombinant DNA segments introduced to genetically modified maize (Zea mays).

Polymerase Chain Reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. In this paper, recombinant DNAs introduced into the seven lines of GM maize, such as Event 176, Bt11, T25, MON810, GA21, DLL25, and MON802, are sequenced. On the basis of the obtained sequence, 14 primer pairs for the detection of the segments, such as promoter, terminator regions, and construct genes, were designed. To confirm the specificities of the designed primer pairs, PCR was performed on genomic DNAs extracted from GM and non-GM maize, GM and non-GM soy, and other cereal crops. Because the presence of the corresponding DNA segments was specifically detected in GM crops by the designed primer pairs, it was concluded that this method is useful for fast and easy screening of GM crops including unauthorized ones.