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Two new methods were developed for identifying Cynaeus angustus (LeConte) (Coleoptera: Tenebrionidae) by DNA amplification using simplex and real-time PCR targeting the cytochrome c oxidase subunit I (COI) sequence reported previously. The specificities of the PCR primers and probe were also confirmed by the two PCR methods using the 22 main stored-product insect species, including DNA samples from nine tenebrionid beetle species. The results showed that the newly developed simplex and real-time PCR-based methods have sufficient specificity for analysis. The limits of detection for C. angustus total DNA by the simplex and multiplex PCR were 320 fg and 20 pg, respectively.

The identification of insect species is generally achieved by analyzing the specific morphological characteristics or, in more recent years, by polymerase chain reaction (PCR) assays of DNA sequences. PCR assays, which detect specific segments of DNA, have been established as a highly sensitive method for a wide range of applications, including the verification of hereditary diseases and for criminal investigations. For the identification of insects, the CO1 (cytochrome c oxidase subunit I) gene of mitochondrial (mt) DNA is often used. However, analyses of the entire mtDNA sequence might yield more reliable regions for use in the identification of insect species. Here we developed a method for real-time PCR identification of the khapra beetle, Trogoderma granarium Everts, by comparing the whole mtDNA sequences of three species of dermestid beetle: the khapra beetle, the varied carpet beetle, Anthrenus verbasci (L.), and the black carpet beetle, Attagenus unicolor japonicus Reitter. Several khapra beetle-specific mtDNA regions were identified and selected for use in the real-time PCR identification of these beetles based on Taq-Man® chemistry. Specificity tests conducted with the DNAs of several insects, including those of the three beetles described above, revealed that the primer/probe sets designed for the real-time PCR were highly specific to their targets.

Here, we report the developed cyclo olefin polymer (COP) microfluidic chip on a fabricated rotating heater stage that utilizes centrifugation-assisted thermal cycle in a ring-structured microchannel for polymerase chain reaction (PCR). The PCR solution could be driven by thermal convection and continuously exchanged high/low temperatures in a ring structured microchannel without the use of typical syringe pump. More importantly, the flow rate was controlled by the relative gravitational acceleration only. The platform enables amplification within 10 min at 5G and has a detection limit of 70.5 pg/channel DNA concentration (β-actin, 295 bp). The current rotating system is capable of testing four different samples in parallel. The microfluidic chip can be preloaded with the PCR premix solution for on-site utility, and, with all of the features integrated to the system, the test can be conducted without the need for specialized laboratory and trained laboratory staff. In addition, this innovative chemical reaction technique has the potential to be utilized in other micromixing applications.

To study impacts of various random effects and parameters of collaborative studies on the precision of quantitation methods of genetically modified (GM) crops, we developed a set of random effects models for cycle time values of a standard curve-based relative real-time PCR that makes use of an endogenous gene sequence as the internal standard. The models and data from a published collaborative study for six GM lines at four concentration levels were used to simulate collaborative studies under various conditions. Results suggested that by reducing the numbers of well replications from three to two, and standard levels of endogenous sequence from five to three, the number of unknown samples analyzable on a 96-well PCR plate in routine analyses could be almost doubled, and still the acceptable repeatability RSD (RSDr < or = 25%) and the reproducibility RSD (RSDR < 35%) of the collaborative study could be met. Further, RSDr and RSD(R) were found most sensitive to random effects attributable to inhomogeneity among blind replicates, but they were little influenced by those attributable to DNA extractions. The proposed models are expected to be useful for optimizing standard curve-based relative quantitation methods for GM crops by real-time PCR and their collaborative studies.

A PCR-based method was developed to distinguish between durum/common wheat and common wheat by leveraging slight differences of DNA sequence in Starch Synthase II (SS II) coded on wheat A, B and D genomes. A primer pair, SS II ex7-U/L, was designed to hybridize with a conserved DNA sequence region found in SS II-A, B and D genes. Another primer pair, SS II-D 1769U/1889L, was constructed to recognize a unique sequence in the SS II-D gene. The target region of SS II ex7-U/L with the size of 114 bp was amplified from durum and common wheat DNA, while no amplification was observed from any cereals other than those in the wheat genus. A DNA fragment with the size of 121 bp was specifically amplified from common wheat with SS II-D 1769U/1889L. In blended flour prepared from wheat and other cereals, the developed PCR system composed of two primer pairs effectively detected durum/common wheat and common wheat. These results suggested that PCR using two primer pairs is useful for detecting common and/or durum wheat in blended flour and could be utilized to ensure accurate food labeling.

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.

Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready® soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.

Novel real-time PCR-based quantitative methods were developed for three GM maize events; MON863, NK603 and TC1507. The quantitative methods were designed to amplify an event-specific segment for MON863 and NK603, and a construct-specific segment for TC1507. We also developed an event-specific quantitative method for T25. The conversion factor (Cf), which is required for calculating the GMO amount, was determined using three types of real-time PCR equipment; the ABI PRISM 7700,7900HT and 7500. The quantitative methods were evaluated by blind testing in an interlaboratory study using the ABI PRISM 7700 and 7900HT, and in a multilaboratory trial using the ABI PRISM 7500. The trueness, precision, and limit of quantitation were determined. Although the biases expressing the trueness for MON863, TC1507, and T25 were slightly high, all the data suggested that the developed methods were suitable for identification and quantification of these GM maize events.

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.

Polypropylene microtubes (tubes) are generally used for bio-material tests in addition to PCR tests such as genetically modified organism (GMO) testings. However, the choice of suitable tubes is quite important, because it might influence the results: DNA binding and/or elution of chemical substances sometimes occurs. In this study, we established methods to select tubes with the most suitable characteristics for DNA testing.

A novel real-time PCR-based analytical method was established for the event-specific quantification of a GM soybean event MON89788. The conversion factor (C(f)) which is required to calculate the GMO amount was experimentally determined. The quantitative method was evaluated by a single-laboratory analysis and a blind test in a multi-laboratory trial. The limit of quantitation for the method was estimated to be 0.1% or lower. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were both less than 20%. These results suggest that the established method would be suitable for practical detection and quantification of MON89788.

A novel multiplex PCR method was developed for simultaneous event-specific detection of four events of GM maize, i.e., DAS-59122-7, MIR604, MON88017, and MON863. The single laboratory examination of analytical performance using simulated DNA mixtures containing GM DNA at various concentrations in non-GM DNA suggested that the limits of detection (LOD) of the multiplex PCR method were 0.16% for MON863, MIR604, and MON88017, and 0.078% for DAS-59122-7. We previously developed a nonaplex (9plex) PCR method for eight events of GM maize, i.e., Bt11, Bt176, GA21, MON810, MON863, NK603, T25, and TC1507. Together with the nonaplex PCR method, the newly developed method enabled the detection and identification of eleven GM maize events that are frequently included in commercial GM seed used in Japan. In addition, this combinational analysis may be useful for the identification of combined event products of GM maize.

A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this.

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.

We developed a novel type of real-time polymerase chain reaction (PCR) array with TaqMan chemistry as a platform for the comprehensive and semiquantitative detection of genetically modified (GM) crops. Thirty primer-probe sets for the specific detection of GM lines, recombinant DNA (r-DNA) segments, endogenous reference genes, and donor organisms were synthesized, and a 96-well PCR plate was prepared with a different primer-probe in each well as the real-time PCR array. The specificity and sensitivity of the array were evaluated. A comparative analysis with the data and publicly available information on GM crops approved in Japan allowed us to assume the possibility of unapproved GM crop contamination. Furthermore, we designed a Microsoft Excel spreadsheet application, Unapproved GMO Checker version 2.01, which helps process all the data of real-time PCR arrays for the easy assumption of unapproved GM crop contamination. The spreadsheet is available free of charge at http://cse.naro.affrc.go.jp/jmano/index.html .

We analyzed the DNA fragments extracted from four rice vermicelli products. The Bacillus thuringiensis (Bt) rice line, which has a construct similar to the GM Shanyou 63 line, was detected in some vermicelli products by identification of the junction region sequence between rice Act1 promoter and the Cry1Ac gene, and that between Cry1Ac and nos. In addition, we also detected a different Bt rice line by means of the junction region sequence between the maize ubiquitin promoter and cry1Ab gene and that between the cauliflower mosaic virus 35S promoter and the hygromycin phosphotransferase in some vermicelli products. Accordingly, we for the first time have detected the two transgenic Bt rice lines contaminating rice vermicelli samples. Furthermore, we developed a duplex real-time polymerase chain reaction (PCR) method for the simultaneous detection of both Bt rice lines. Keywords: Genetically modified rice; Bt toxin; detection method; real-time PCR; rice vermicelli; Bacillus thuringiensis

An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (muTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the "initial start-up" in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with muTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.

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.

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.

Transglucosylation activities of spinach alpha-glucosidase I and IV, which have different substrate specificity for hydrolyzing activity, were investigated. In a maltose mixture, alpha-glucosidase I, which has high activity toward not only maltooligosaccharides but also soluble starch and can hydrolyze isomaltose, produced maltotriose, isomaltose, and panose, and alpha-glucosidase IV, which has high activity toward maltooligosaccharides but faint activity toward soluble starch and isomaltose, produced maltotriose, kojibiose, and 2,4-di-alpha-D-glucosyl-glucose. Transglucosylation to sucrose by alpha-glucosidase I and IV resulted in the production of theanderose and erlose, respectively, showing that spinach alpha-glucosidase I and IV are useful to synthesize the alpha-1,6-glucosylated and alpha-1,2- and 1,4-glucosylated products, respectively.

A cDNA encoding spinach alpha-glucosidase was cloned and sequenced by the reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA comprised 2867 bp, and included an open reading frame which encodes a polypeptide of 903 amino acid residues. The calculated molecular mass of 101 kDa was larger than those of native alpha-glucosidases in spinach seeds, which are 78, 78, 82, and 82 kDa by SDS-PAGE for alpha-glucosidase I, II, III, and IV, respectively. The deduced amino acid sequence included those of tryptic peptides from native enzymes. Southern blot analysis suggested that the alpha-glucosidase gene was a single-copy gene. These results indicate the possibility that the multiplicity of alpha-glucosidase in spinach occurs via post-translational modification.

Qualitative PCR methods for the genetically modified (GM) maize events MON810, Bt11, and GA21, and the 35S promoter (P35S) region of the cauliflower mosaic virus (CaMV) were evaluated in an interlaboratory study. Real-time PCR-based quantitative methods for these GM events using the same primer pairs had already been validated in previous studies. Fifteen laboratories in Japan participated in this interlaboratory study. Each participant extracted DNA from blind samples, performed qualitative PCR assays, and then detected the PCR products with agarose gel electrophoresis. The specificity, sensitivity, and false-negative and false-positive rates of these methods were determined with different concentrations of GM mixing samples. LODs of these methods for MON810, Bt11, GA21, and the P35S segment calculated as the amount of MON810 were 0.2, 0.2, 0.1, and 0.2% or less, respectively, indicating that the LODs of MON810, Bt11, and P35S were lower than 10 copies, and the LOD of GA21 was lower than 25 copies of maize haploid genome. The current study demonstrated that the qualitative methods would be fit for the detection and identification of these GM maize events and the P35S segment.

Four molecular forms of α-glucosidase were isolated from spinach seeds by several kinds of chromatography. The molecular masses of α-glucosidases I, II, III, and IV were 78, 78, 82, and 82 kDa by SOS–PAGE, and 62, 62, 190, and 70 kDa by gel filtration, respectively. α-Glucosidases I and II showed similar enzymatic properties, in which the Km for soluble starch was about 10 times lower than that for maltose, and they had higher activity not only toward malto-oliosaccharides but also toward α-glucans. The optimum pH was 4.5–5.5 and about 50% of the activity remained after incubation at 70°C for 20 min. On the other hand, α-glucosidases III and IV showed similar enzymatic properties, in which the Km for maltose was 3–4 times lower than that for soluble starch, and they had high activity toward malto-oligosaccharides but faint activity toward α-glucans. The optimum pH was 4.5–5.0 and no activity was found after incubation at 70°C for 20 min. However, anti-α-glucosidase III serum formed precipitation specifica...

To develop a method for detecting GM wheat that may be marketed in the near future, we evaluated the proline-rich protein (PRP) gene as an endogenous reference gene of common wheat (Triticum aestivum L.) and durum wheat (Triticum durum L.). Real-time PCR analysis showed that only DNA of wheat was amplified and no amplification product was observed for phylogenetically related cereals, indicating that the PRP detection system is specific to wheat. The intensities of the amplification products and Ct values among all wheat samples used in this study were very similar, with no nonspecific or additional amplification, indicating that the PRP detection system has high sequence stability. The limit of detection was estimated at 5 haploid genome copies. The PRP region was demonstrated to be present as a single or double copy in the common wheat haploid genome. Furthermore, the PRP detection system showed a highly linear relationship between Ct values and the amount of plasmid DNA, indicating that an appropriate calibration curve could be constructed for quantitative detection of GM wheat. All these results indicate that the PRP gene is a suitable endogenous reference gene for PCR-based detection of GM wheat.

We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports.

To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

The Bt11 maize-specific qualitative detection method based on polymerase chain reaction (PCR) in the JAS analytical test handbook has been widely used for administrative monitoring of GM crops and quality control of commercially distributed grains. In the present investigation, some apparently false-positive detections were observed in assays using the Bt11 maize-specific method, and these erroneous results were proved to have been caused by non-specific DNA amplification. We improved the detection method to reduce non-specific amplification by decreasing the concentration of magnesium ions in the PCR mixture. The subsequent evaluation of analytical performance demonstrated no marked difference between the currently used and the improved methods, except for the reduced non-specific amplification. We conclude that the currently used standard method should be replaced with the improved method for the reliable detection of Bt11 maize.

To screen for unauthorized genetically modified organisms (GMO) in the various crops, we developed a multiplex real-time polymerase chain reaction high-resolution melting-curve analysis method for the simultaneous qualitative detection of 35S promoter sequence of cauliflower mosaic virus (35SP) and the nopaline synthase terminator (NOST) in several crops. We selected suitable primer sets for the simultaneous detection of 35SP and NOST and designed the primer set for the detection of spiked ColE1 plasmid to evaluate the validity of the polymerase chain reaction (PCR) analyses. In addition, we optimized the multiplex PCR conditions using the designed primer sets and EvaGreen as an intercalating dye. The contamination of unauthorized GMO with single copy similar to NK603 maize can be detected as low as 0.1% in a maize sample. Furthermore, we showed that the present method would be applicable in identifying GMO in various crops and foods like authorized GM soybean, authorized GM potato, the biscuit which is contaminated with GM soybeans and the rice which is contaminated with unauthorized GM rice. We consider this method to be a simple and reliable assay for screening for unauthorized GMO in crops and the processing food products.

DNA analysis with the polymerase chain reaction (PCR) has become a routine part of medical diagnostics, environmental inspections, food evaluations, and biological studies. Furthermore, the development of a microscale PCR chip is an essential component of studies aimed at integrating PCR into a micro total analysis system (mu-TAS). However, the occurrence of air bubbles in microchannels complicates this process. In this study, we investigated a new technique based on the fluid dynamics of laminar flow that utilizes a small amount of mineral oil at the beginning of sample injection to prevent air bubbles from occurring in microchannels. We also further optimized the pressure, the length of the pressurizing channel and the volume of oil, thus making our microfluidic device more useful for high-temperature PCR. Additionally, quantitative continuous-flow PCR was performed using the optimized PCR chip in order to detect genetically modified (GM) maize. DNA was extracted from GM maize, MON 810, and non-GM maize at several concentrations from 0% (w/v) to 100% (w/v). The DNA amplification signals were then analyzed on the PCR chip using a laser-based system. The signal from our microfluidic PCR chip was found to increase in direct proportion to the initial GM maize concentration.

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

In this study, we are reporting for the first time an efficient, accurate and inexpensive rapid detection system which employs the integration of isothermal amplification and subsequent analysis of unpurified amplicons by an electrochemical system. In our experiments, loop-mediated isothermal amplification (LAMP) with its higher efficiency than PCR was performed at a constant temperature (65 degrees C). Amplification products were combined with a redox active molecule Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)] and analyzed by a DNA stick (DS) which is integrated with a disposable electrochemical printed (DEP) chip using linear sweep voltammetry (LSV). The DNA minor groove binding of the H33258 molecule causes a significant drop in the peak current intensity of the H33258 oxidation. The phenomenon of DNA binding induced by H33258, in addition to changes in the anodic current peak, was used to detect maize CBH 351 variety (StarLink). Since laborious probe immobilization was not required, and amplification and detection were performed on a single device, our biosensor eliminates potential cross-contamination. We believe that this type of sensor will have an unprecedented impact for environmental protection.

We developed a multiplex polymerase chain reaction (PCR)-multiplex ligase chain reaction (LCR) (MPCR-MLCR) technique as a novel approach for the simultaneous detection of recombinant DNA segments (e.g., promoters, trait genes, and terminators) of genetically modified (GM) crops. With this technique, target DNA regions were amplified by multiplex PCR, the PCR products were then subjected to multiplex LCR as template DNAs, and the LCR products were then analyzed by polyacrylamide gel electrophoresis and subsequent fluorescent scanning. Seven recombinant DNA segments commonly introduced into some GM crop lines were selected as target DNA regions. In addition, another MPCR-MLCR system for the simultaneous detection of three endogenous DNA segments was designed as a positive control test. The specificity and sensitivity of the method were examined. The method allowed us to detect GM crops comprehensively and is expected to be utilized for efficient screening of GM crops into which any one of the seven recombinant DNA segments have been introduced, and for profiling the segments.

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.

A real-time PCR detection method was developed for event-specific quantitation of Roundup Ready maize, GA21. The developed PCR method was designed to amplify an artificial junction site between the native maize genome DNA and the recombinant DNA of GA21 maize, which provides only one target sequence per haploid of GA21 genome. Thus, the amplification efficiency of the event-specific target for GA21 became closely similar to the amplification of SSIIb, and the conversion factor (Cf) for the quantitation method was similar to the theoretical value. The developed method demonstrated better performance than the existing construct-specific method that has been used as a Japanese official method. The developed method can easily be combined with the real-time PCR targeting of the CaMV35S promoter, and the multiplexed method should be an effective screening method for GM maize.

In many countries, the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved GM varieties. The GMO content in a maize sample containing the combined-trait (stacked) GM maize as determined by the currently available methodology is likely to be overestimated. However, there has been little information in the literature on the mixing level and varieties of stacked GM maize in real sample grains. For the first time, the GMO content of non-identity-preserved (non-IP) maize samples imported from the United States has been successfully determined by using a previously developed individual kernel detection system coupled to a multiplex qualitative PCR method followed by multichannel capillary gel electrophoresis system analysis. To clarify the GMO content in the maize samples imported from the United States, determine how many stacked GM traits are contained therein, and which GM trait varieties frequently appeared in 2005, the GMO content (percent) on a kernel basis and the varieties of the GM kernels in the non-IP maize samples imported from the United States were investigated using the individual kernel analysis system. The average (± standard deviation) of the GMO contents on a kernel basis in five non-IP sample lots was determined to be 51.0 ± 21.6%, the percentage of a single GM trait grains was 39%, and the percentage of the stacked GM trait grains was 12%. The MON810 grains and NK603 grains were the most frequent varieties in the single GM traits. The most frequent stacked GM traits were the MON810 x NK603 grains. In addition, the present study would provide the answer and impact for the quantification of GM maize content in the GM maize kernels on labeling regulation.

We developed a specific method to extract DNA from rice grain samples and modified the qualitative real-time PCR method provided by Bayer Co., Ltd. for reliable detection of the genetically modified (GM) rice variety, LLRice601, which has not undergone safety assessment for regulatory approval in Japan. Moreover, we conducted a data analysis to confirm the results obtained with real-time PCR. The yields of DNA extracted from powdered samples of rice grains were almost equal among 5 different varieties of rice, and there was no significant difference in the yield over three days. Reliable results were obtained using 50 ng of the extracted DNA as the template for real-time PCR. To examine the adequacy of the methods, we organized an interlaboratory study with the participation of 2 laboratories, in which 80 test samples were analyzed in a blinded manner. The statistical analysis revealed no significant difference in the Ct value for the endogenous gene of the DNA samples and for the targeted DNA sequence of 0.1% samples. The limit of detection of the method was approximately 0.1%. Analysis of the fluorescence intensity of the PCR-amplified product of the construct-specific DNA sequence suggested that it may be reasonable to judge a sample as positive when a Ct value of less than 40 is obtained.

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.

A cDNA encoding spinach putative phospholipid hydroperoxide glutathione peroxidase (PHGPX) was cloned and sequenced. The cDNA included an open reading frame that encoded a polypeptide of 171 amino acid residues. The deduced amino acid sequence showed about 77 and 50% similarity to plant putative PHGPXs and mammalian PHGPXs, respectively. PCR products with the same size as that of the spinach putative PHGPX were obtained from maize, soybeans, and Arabidopsis, suggesting the expression of putative PHGPX genes in other plants.

The rate parameters for maltooligosaccharides of two α-glucosidases from spinach seeds were examined and subsite affinities were evaluated. The subsite affinities for subsites 1, 2, 3, 4, 5, 6, and 7 in the active site of α-glucosidase A and B were 1.73, 2.91, 1.10, 0.25, 0.94, 0.03, and −0.01 kcal/mol, and 0.33, 4.94, 0.26, 0.14, −0.24, −0.52, and 0.14 kcal/mol, respectively. The six and four subsites exist in α-glucosidase A and B, respectively, which maltooligosaccharides or soluble starch could be bound.