Development and Validation of a New Robust Detection Method for Low-Content DNA Using ΔΔCq-Based Real-Time PCR with Optimized Standard Plasmids as a Control Sample.

Real-time polymerase chain reaction (PCR) is the gold standard for DNA detection in many fields, including food analysis. However, robust detection using a real-time PCR for low-content DNA samples remains challenging. In this study, we developed a robust real-time PCR method for low-content DNA using genetically modified (GM) maize at concentrations near the limit of detection (LOD) as a model. We evaluated the LOD of real-time PCR targeting two common GM maize sequences (P35S and TNOS) using GM maize event MON863 containing a copy of P35S and TNOS. The interlaboratory study revealed that the LOD differed among laboratories partly because DNA input amounts were variable depending on measurements of DNA concentrations. To minimize this variability for low-content DNA samples, we developed ΔΔCq-based real-time PCR. In this study, ΔCq and ΔΔCq are as follows: ΔCq = Cq (P35S or TNOS) - Cq (SSIIb; maize endogenous gene), ΔΔCq = ΔCq (analytical sample) - ΔCq (control sample at concentrations near the LOD). The presence of GM maize was determined based on ΔΔCq values. In addition, we used optimized standard plasmids containing SSIIb, P35S, and TNOS with ΔCq equal to the MON863 genomic DNA (gDNA) at concentrations near the LOD as a control sample. A validation study indicated that at least 0.2% MON863 gDNA could be robustly detected. Using several GM maize certified reference materials, we have demonstrated that this method was practical for detecting low-content GM crops and thus for validating GM food labeling. With appropriate standards, this method would be applicable in many fields, not just food.

Development and Interlaboratory Validation of a Novel Reproducible Qualitative Method for GM Soybeans Using Comparative Cq-Based Analysis for the Revised Non-GMO Labeling System in Japan.

Many countries have implemented the labeling system of genetically modified organisms (GMO). In Japan, the regulatory threshold for non-GMO labeling will be revised and restricted to undetectable by April 2023. The practical criterion for the revised system is based on the limit of detection (LOD). However, determining whether the commingling of GMO levels exceeds the LOD is challenging because GM contents close to the LOD are usually below the limit of quantification. In this study, we developed a qualitative method based on comparative Cq-based analysis targeting cauliflower mosaic virus 35S promoter and GM soybean MON89788 event-specific sequences that could be applicable to the revised non-GMO labeling. ΔCq values between the target and endogenous sequences were calculated, and the ΔΔCq value obtained was used as a criterion to determine analytical samples with GM contents exceeding the threshold. To improve the reproducibility of the method, we used a standard plasmid that yields equivalent and stable ΔCq values comparable with those obtained from LOD samples. The developed method was validated with an interlaboratory study. The new qualitative detection concept would be useful for ensuring robust and reproducible results among laboratories, particularly for detecting low-copy-number DNA samples.

[Evaluation of a Species-specific, Stable, and Endogenous Sequence of Eggplant (Solanum melongena) using LAMP for the Detection of Genetically Modified Eggplants].

Species-specific endogenous reference sequences are indispensable in the development of methods to detect genetically modified (GM) crops for food and feed. We analyzed a partial sequence derived from the ß-fructosidase gene among several solanaceous species and developed a new eggplant specific detection method using loop-mediated isothermal amplification (LAMP). LAMP is a rapid, specific, and cost-effective technique. The species-specificity and stability of the developed method were evaluated using 18 eggplant cultivars and other crops including solanaceous plants. The limit of detection was also evaluated. The developed method showed high specificity for eggplants and stability among the eggplant cultivars tested. These results suggested that the developed method would be useful as a positive control for the detection of GM eggplants with LAMP.

Development and Testing of an Individual Kernel Detection System for Genetically Modified Soybean Events in Non-identity-preserved Soybean Samples.

A genetically modified (GM) soybean kernel detection system using combination of DNA preparation from individual soybean kernels and event-specific real-time PCR was developed to simultaneously identify GM soybean events authorized for food after safety assessments in Japan. Over 100 kernels in the non-identity-preserved soybean samples imported from the United States of America (two U.S.A. lots) and Brazil (one lot) were randomly selected and examined. In total, 98 and 96% of the two independent U.S.A. lots, and 100% of the Brazilian lot contained GM soybean kernels. Herbicide-tolerant events, MON89788 (trade name Genuity® Roundup Ready 2 Yield™), GTS 40-3-2 (trade name Roundup Ready™ soybean) and A2704-12 (trade name Liberty Link® soybean), were detected similarly in both U.S.A. lots. In the Brazilian lot, in addition to GTS 40-3-2, a stacked GM event, MON87701 × MON89788, having insect-resistance and herbicide-tolerance, was detected. There were no unauthorized GM soybeans comingled, and the ratio of GM soybean events detected was consistent with statistical reports on the cultivated GM soybean events in both countries.

Detection of 30 bp DNA fragments with a sensitive modified Southern blot analysis.

To evaluate crops generated by new breeding techniques, it is important to confirm the removal of recombinant DNAs (rDNAs) derived from foreign genes including unintentionally introduced short rDNA(s). We attempted to develop a sensitive detection method for such short rDNAs using Southern blot analysis and performed a model study targeting single-copy endogenous genes in plants. To increase the detection sensitivity, the general protocol for Southern blot analysis was modified. In the model study, we used endogenous-gene-targeting probes in which complementary sequences were serially replaced by dummy sequences, and detected complementary sequences as well as 30 bp. We further evaluated the sensitivity using short rDNAs derived from GM sequences as pseudoinsertions, and the results demonstrated that rDNA-insertions as small as 30 bp could be detected. The results suggested that unintentionally introduced rDNA-insertions were 30 bp or more in length could be detected by the Southern blot analysis.

Rapid DNA template preparation directly from a rice sample without purification for loop-mediated isothermal amplification (LAMP) of rice genes.

Rapid DNA template preparation directly from a single rice (Oryza sativa) grain or rice flour of its equivalent weight was developed for loop-mediated isothermal amplification (LAMP). LAMP efficiency using DNA extract obtained from consecutive addition of alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA) and neutralizing reagent (40 mM Tris-HCl [pH 5]) was comparable to that using an equivalent amount of purified DNA as template. The stability of the prepared DNA extract was confirmed for up to six-day storage at room temperature. Without using any special laboratory devices, the developed method enabled a rapid, simple, and low-cost DNA template preparation method for reliable LAMP testing to detect rice genes.

[Evaluation of Conversion Factors for Genetically Modified Maize Quantification].

To quantify the amount of authorized GM maize or soybean, conversion factor (Cf) values are required for converting the copy number ratio of GM sequence to an endogenous sequence into weight-based GMO amounts. Cf values are available for the several latest real-time PCR instruments such as QuantStudio5, QuantStudio12K Flex, LightCycler 96, and LightCycler 480 for GM soybeans but not for GM maize. For the quantification of GM maize, we experimentally determined the Cf values targeting Cauliflower mosaic virus 35S promoter (P35S), GA21 construct specific, MIR604 event specific and MIR162 event specific sequences using the four real-time PCR instruments.

Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA.

Nucleic acid amplification methods, such as polymerase chain reaction (PCR), are extensively used in many applications to detect target DNA because of their high sensitivity, good reproducibility, and wide dynamic range of quantification. However, analytical quality control when detecting low copy number target DNA is often missing because of a lack of appropriate reference materials. Recent advances in analytical sciences require a method to accurately quantify DNA at the single molecule level. Herein, we have developed a novel method to produce reference material containing a defined copy number of target DNA (referred to as "cell number-based DNA reference material"). In this method, a suspension of cells carrying a single target DNA sequence was ejected by an inkjet head, and the number of cells in each droplet was counted using highly sensitive cameras. The resulting solutions contained a defined copy number of target DNA and could be used as reference materials. The use of the newly developed reference material was compared with that of diluted solutions of target DNA to evaluate the performance of qualitative real-time PCR in terms of the limit of detection (LOD). Our results demonstrated that cell number-based DNA reference material provides more accurate information regarding performance quality. The reference material produced by this method is a promising tool to evaluate assay performance.

[Detection of DNA in Highly Processed Foods].

Highly processed foods, including soy sauce, cornflakes, starch sugar, beet sugar and vegetable oil, are not currently subject to genetically modified (GM) food labeling, because DNA could not be detected in these food products. Here we re-examined the method of DNA extraction from starch syrup, beet sugar and vegetable oil using commercially available DNA extraction kits. We found that DNA was not stably detected by PCR targeting a species-specific endogenous plant gene. The reason for this may have been that the DNA yield was below the detection limit, because PCR inhibition was not observed.

A set of tetra-nucleotide core motif SSR markers for efficient identification of potato (Solanum tuberosum) cultivars.

Simple sequence repeat (SSR) is a popular tool for individual fingerprinting. The long-core motif (e.g. tetra-, penta-, and hexa-nucleotide) simple sequence repeats (SSRs) are preferred because they make it easier to separate and distinguish neighbor alleles. In the present study, a new set of 8 tetra-nucleotide SSRs in potato (Solanum tuberosum) is reported. By using these 8 markers, 72 out of 76 cultivars obtained from Japan and the United States were clearly discriminated, while two pairs, both of which arose from natural variation, showed identical profiles. The combined probability of identity between two random cultivars for the set of 8 SSR markers was estimated to be 1.10 × 10-8, confirming the usefulness of the proposed SSR markers for fingerprinting analyses of potato.

Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens.

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14-16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

Development of a reference material of a single DNA molecule for the quality control of PCR testing.

We developed a reference material of a single DNA molecule with a specific nucleotide sequence. The double-strand linear DNA which has PCR target sequences at the both ends was prepared as a reference DNA molecule, and we named the PCR targets on each side as confirmation sequence and standard sequence. The highly diluted solution of the reference molecule was dispensed into 96 wells of a plastic PCR plate to make the average number of molecules in a well below one. Subsequently, the presence or absence of the reference molecule in each well was checked by real-time PCR targeting for the confirmation sequence. After an enzymatic treatment of the reaction mixture in the positive wells for the digestion of PCR products, the resultant solution was used as the reference material of a single DNA molecule with the standard sequence. PCR analyses revealed that the prepared samples included only one reference molecule with high probability. The single-molecule reference material developed in this study will be useful for the absolute evaluation of a detection limit of PCR-based testing methods, the quality control of PCR analyses, performance evaluations of PCR reagents and instruments, and the preparation of an accurate calibration curve for real-time PCR quantitation.

Identification and detection of genetically modified papaya resistant to papaya ringspot virus strains in Thailand.

Many lines of genetically modified (GM) papaya (Carica papaya Linnaeus) have been developed worldwide to resist infection from various strains of papaya ringspot virus (PRSV). We found an unidentified and unauthorized GM papaya in imported processed papaya food. Transgenic vector construct that provides resistance to the PRSV strains isolated in Thailand was detected. An original and specific real-time polymerase chain reaction method was generated to qualitatively detect the PRSV-Thailand-resistant GM papaya.

Development and Performance Evaluation of a New Test Kit for Quantifying the Degree of DNA Fragmentation.

BACKGROUND: PCR-based genetic testing of agricultural products and foods is widely used for detecting various analytical targets such as genetically modified organisms and food allergens. However, it is difficult to obtain accurate genetic testing results from processed foods because DNA is fragmented by heat and pressure during food processing. Thus we previously developed an analytical method to quantitatively evaluate the degree of DNA fragmentation for the purpose of quality control of genetic testing for processed foods. OBJECTIVE: Our previous analytical method requires four PCR primer sets, resulting in high reagent costs and heavy analytical workloads. Therefore, we attempted to develop an easy-to-use test kit for quantifying the degree of DNA fragmentation and to evaluate its analytical performance. METHODS: To simplify the analysis procedure, we used only two primer sets. In addition, no-fragmentation control templates were prepared to obtain stable measurement results. The precision of the simplified analysis was evaluated through blind tests among laboratories. RESULTS: It was confirmed that plant species and extracted DNA concentrations had little effect on analysis with the newly developed test kit. In addition, the analytical values indicating the degree of DNA fragmentation exhibited small variability among laboratories. CONCLUSION: We confirmed the high practicality of the developed test kit. Because DNA fragmentation in cells is a universal phenomenon, we anticipate that the test kit will be used not only for quality control of genetic testing but also for food testing, medical diagnostics and other applications in a range of fields. HIGHLIGHTS: The newly developed test kit enables quantitative evaluation of the degree of DNA fragmentation in a simple manner.

Development and interlaboratory validation of quantitative polymerase chain reaction method for screening analysis of genetically modified soybeans.

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.

Evaluation of extraction solutions for biochemical analyses of the proteins in rice grains.

The influence of different extraction solutions on the proteins extracted from rice grains was investigated. The largest amounts of salt-soluble proteins were extracted with solutions supplemented with Tris-HCl at pH 8.0. Rice allergens were analyzed by multiplex immunodetection. Except for α-globulin extracted with the solutions at pH 8.0, which showed a low-molecular-weight band besides the main band, no significant solution-dependent difference among the allergens was found. Total proteins were extracted with four kinds of solution. The extraction of the basic subunit of glutelin was found to be SDS-dependent, and more protein was obtained with extraction solutions supplemented with SDS. The contents of α-globulin and α-amylase/trypsin inhibitors were higher in the extracts without SDS than with SDS. We conclude from the present data that, in order to obtain comparable data from rice grain salt-soluble and total protein analyses, differences in the protein extraction efficiency of solutions used should be taken into consideration.

Effect of gamma irradiation on soybean allergen levels.

The levels of food allergens in gamma-irradiated soybean (0, 2.5, 5, 7.5, 10, 20, and 30 kGy) were investigated by immunoblotting and ELISA, using allergen-specific antibodies and patient serum. After 3 months of storage, Coomassie brilliant blue (CBB) staining indicated similar total protein profiles among the treatments, but that some proteins were degraded by irradiation at high doses. Immunoblotting with specific antibodies for major soybean allergens (ß-conglycinin, Gly m Bd 30 K, soybean trypsin inhibitor, and Gly m 4) resulted in apparent band profiles and intensities that were not significantly changed by irradiation. Competitive inhibition ELISA analyses suggested that there were no significant changes in the allergen contents, except for a decrease in the soybean trypsin inhibitor. The patient IgE binding allergenic protein patterns were not changed by irradiation up to 30 kGy. ELISA using patient serum also revealed that the IgE reactivity to the irradiated soybean extract did not increase from the level of the control, but that the reactivity to some patient serum IgE was significantly decreased by irradiation.

Simulation of collaborative studies for real-time PCR-based quantitation methods for genetically modified crops.

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.

Interlaboratory study of qualitative PCR methods for genetically modified maize events MON810, bt11, GA21, and CaMV P35S.

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.