Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes

With the development of transgenic crops, many countries have issued regulations to label the genetically modified organisms (GMOs) and their derived products. Polymerase Chain Reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods generally need to amplify the transgene and compare the amplified result with that of the corresponding reference gene to obtain reliable results. In this article, we reported the development of specific primers and probe for the rice (Oryza sativa) sucrose phosphate synthase (SPS) gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 13 different rice varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other species, such as wheat, maize, barley, tobacco, soybean, rapeseed, tomato, sunflower, carrot, pepper, eggplant, lupine, mung bean, plum, and Arabidopsis thaliana, were used as templates, which demonstrated that this system was specific for rice. In addition, the results of the Southern blot analysis confirmed that the SPS gene was a single copy in the tested rice varieties. In qualitative and quantitative PCR analyses, the detection sensitivities were 0.05 and 0.005 ng of rice genomic DNA, respectively. To test the practical use of this SPS gene as an endogenous reference gene, we have also quantified the beta-glucuronidase (GUS) gene in transgenic rice using this reference gene. These results indicated that the SPS gene was species specific, had one copy number, and had a low heterogeneity among the tested cultivars. Therefore, this gene could be used as an endogenous reference gene of rice and the optimized PCR systems could be used for practical qualitative and quantitative detection of transgenic rice.     

International collaborative study of the endogenous reference gene LAT52 used for qualitative and quantitative analyses of genetically modified tomato

One tomato ( Lycopersicon esculentum) gene, LAT52, has been proved to be a suitable endogenous reference gene for genetically modified (GM) tomato detection in a previous study. Herein are reported the results of a collaborative ring trial for international validation of the LAT52 gene as endogenous reference gene and its analytical systems; 14 GMO detection laboratories from 8 countries were invited, and results were finally received from 13. These data confirmed the species specificity by testing 10 plant genomic DNAs, less allelic variation and stable single copy number of the LAT52 gene, among 12 different tomato cultivars. Furthermore, the limit of detection of LAT52 qualitative PCR was proved to be 0.1%, which corresponded to 11 copies of haploid tomato genomic DNA, and the limit of quantification for the quantitative PCR system was about 10 copies of haploid tomato genomic DNA with acceptable PCR efficiency and linearity. Additionally, the bias between the test and true values of 8 blind samples ranged from 1.94 to 10.64%. All of these validated results indicated that the LAT52 gene is suitable for use as an endogenous reference gene for the identification and quantification of GM tomato and its derivates.     

A rapeseed-specific gene, acetyl-CoA carboxylase, can be used as a reference for qualitative and real-time quantitative PCR detection of transgenes from mixed food samples

Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA.     

Evaluation of four genes in rice for their suitability as endogenous reference standards in quantitative PCR

The genetically modified (GM) food/feed quantification depends on the reliable detection systems of endogenous reference genes. Currently, four endogenous reference genes including sucrose phosphate synthase (SPS), GOS9, phospholipase D (PLD), and ppi phosphofructokinase (ppi-PPF) of rice have been used in GM rice detection. To compare the applicability of these four rice reference genes in quantitative PCR systems, we analyzed the target nucleotide sequence variation in 58 conventional rice varieties from various geographic and phylogenic origins, also their quantification performances were evaluated using quantitative real-time PCR and GeNorm analysis via a series of statistical calculation to get a "M value" which is negative correlation with the stability of genes. The sequencing analysis results showed that the reported GOS9 and PLD taqman probe regions had detectable single nucleotide polymorphisms (SNPs) among the tested rice cultivars, while no SNPs were observed for SPS and ppi-PPF amplicons. Also, poor quantitative performance was detectable in these cultivars with SNPs using GOS9 and PLD quantitative PCR systems. Even though the PCR efficiency of ppi-PPF system was slightly lower, the SPS and ppi-PPF quantitative PCR systems were shown to be applicable for rice endogenous reference assay with less variation among the C(t) values, good reproducibility in quantitative assays, and the low M values by the comprehensive quantitative PCR comparison and GeNorm analysis.     

Novel reference gene, PKABA1, used in a duplex real-time polymerase chain reaction for detection and quantitation of wheat- and barley-derived DNA

We report the development of a duplex real-time Polymerase Chain Reaction (PCR) for the simultaneous detection and quantification of wheat- and barley-derived DNA. We used a single primer pair to amplify the single-copy gene PKABA1 from wheat and barley, using minor-groove-binding probes to distinguish between the two cereals. The assay was fully specific, and different wheat and barley cultivars exhibited similar Ct values, indicating stability across cultivars with respect to allelic and copy number composition. The limits of detection were 5 and 10 PCR-forming units for wheat and barley, respectively, making the duplex assay as sensitive as other singleplex reference gene systems published. We were able to detect both wheat and barley simultaneously in real food samples, and the duplex assay is considered to be suitable as an endogenous reference gene system for the detection and quantification of wheat and barley in genetically modified organisms (GMO) and other food and feed analyses.     

Event-specific qualitative and quantitative PCR detection methods for transgenic rapeseed hybrids MS1xRF1 and MS1xRF2

Except for the events RT73, MS8, RF3, and T45, event-specific detection methods for most commercialized genetically modified (GM) rapeseed varieties have not been established, and as a result, the enforcement of genetically modified organism labeling policies has been hindered. The genetically modified rapeseeds, MS1xRF1 and MS1xRF2, are 2 of 11 approved GM-rapeseed varieties for commercialization. In this study, the right border junction fragments between the gene construct and the rapeseed genome of events RF1, RF2, and MS1 were isolated using the commercially available GenomeWalker technology. Homology analysis indicated that the gene construct of RF1 integrated upstream of the nuclease gene, and that of the RF2 and MS1 inserted into the exon region of a gene encoding for an unknown protein. The event-specific primer pairs and corresponding probes were designed on the basis of the revealed right border junction fragments. Then, we successfully developed the identification and quantification methods for the gene-stacked hybrids MS1xRF1 and MS1xRF2 using those primers and probes. The relative limit of detection in the qualitative polymerase chain reaction (PCR) was 0.013% for the RF2 and MS1 assays using 100 ng of rapeseed DNA per reaction and 0.13% for the RF1 assay. The absolute limit of detection in the quantitative PCR was approximately one to two initial copies for each of the three event-specific assays. The evaluation of the real-time PCR assays revealed that the qualitative and quantitative methods developed by focusing on the gene-stacked hybrids MS1xRF1 and MS1xRF2 were highly specific, sensitive, and suitable for samples with a low quantity of DNA.     

Use of quantitative real-time PCR to estimate maize endogenous DNA degradation after cooking and extrusion or in food products

Polymerase chain reaction (PCR) is being used increasingly to detect DNA sequences for food quality testing for GM content, microbial contamination, and ingredient content. However, food processing often results in DNA degradation and therefore may affect the suitability of PCR or even DNA sequence detection for food quality assurance. This paper describes a novel approach using quantitative real-time PCR (qPCR) to estimate the extent of DNA degradation. With use of two maize endogenous nuclear sequences, sets of four qPCR assays were developed to amplify target sequences ranging from<100 bp to approximately 1000 bp. The maize nuclear sequences used encode chloroplastic glyceraldehyde-3-phosphate dehydrogenase and cell wall invertase. The utility of the qPCR approach for quantifying the effective concentration of maize DNA that is needed to amplify variable length DNA sequences was demonstrated using samples of maize cornmeal cooked in water for variable times, extrusion products developed using different barrel temperature and torque settings, and a range of food products from supermarket shelves. Results showed that maize DNA was substantially degraded by a number of processing procedures, including cooking for 5 min or more, extrusion at high temperatures and/or high torque settings, and in most processed foods from supermarket shelves. Processing also reduced the effective concentration of DNA sequences capable of directing amplification of the <100 bp assays as well, particularly after popping of popping corn or extrusion at a combination of high temperature and torque settings. The approach for quantifying DNA degradation described in this paper may also be of use in disciplines where understanding the extent of DNA degradation is important, such as in environmental, forensic, or historical samples.     

Selection of reference genes for quantitative real-time PCR analysis in Lathyrus sativus L. under different development stages and drought stress

Genetic Resources and Crop Evolution - Grass pea (Lathyrus sativus L.) is a robust legume crop with high protein content and good stress tolerance. However, it needs genetic improvement due to the...     

Qualitative and quantitative polymerase chain reaction assays for an alfalfa (Medicago sativa)-specific reference gene to use in monitoring transgenic cultivars

Genetically modified (GM) alfalfa (Medicago sativa) was marketed for the first time in 2005. For countries with established thresholds for GM plants, methods to detect and quantify their adventitious presence are required. We selected acetyl CoA carboxylase as a reference gene for the detection and quantification of GM alfalfa. Two qualitative polymerase chain reaction (PCR) assays (Acc1 and Acc2) were designed to detect alfalfa. Both were specific to alfalfa, amplifying DNA from 12 separate cultivars and showing negative results for PCR of 15 nonalfalfa plants. The limits of detection for Acc1 and Acc2 were 0.2 and 0.01%, respectively. A quantitative real-time PCR assay was also designed, having high linearity (r > 0.99) over alfalfa standard concentrations ranging from 100 to 2.0 x 10(5) pg of alfalfa DNA per PCR. The real-time PCR assay was effective in quantifying alfalfa DNA from forage- and concentrate-based mixed diets containing different amounts of alfalfa meal.     

Pea detection in food and feed samples by a real-time PCR method based on a specific legumin gene that allows diversity analysis

Real-time polymerase chain reaction is currently being used for the identification and quantification of plant and animal species as well as microorganisms in food or feed samples based on the amplification of specific sequences of low copy genes. We report here the development of a new real-time PCR method for the detection and quantification of the pea (Pisum sativum) based on the amplification of a specific region of the legS gene. The specificity was evaluated in a wide range of plant species (51 varieties of Pisum sp., and 32 other plant species and varieties taxonomically related or nonrelated). The method allows the detection and quantification of as low as 21.6 pg of DNA, which corresponds to 5 haploid genome copies. The system has been shown to be sensitive, reproducible and 100% specific for the rapid detection and quantification of pea DNA in processed food and feed samples, being therefore suitable for high-throughput analysis.     

Comparison of real-time PCR detection chemistries and cycling modes using Mon810 event-specific assays as model

The most widely accepted methods for accurate quantitative detection of genetically modified organisms rely on real-time PCR. Various detection chemistries are available for real-time PCR. They include sequence-unspecific DNA labeling dyes such SYBR-Green I and the use of both universal (e.g., AmpliFluor) and sequence-specific double-labeled probes, the latter comprising hybridization (e.g., Molecular Beacon) and hydrolysis (e.g., TaqMan or MGB) probes. Also, new real-time PCR devices and reagents allowing fast cycling reactions exist. Five Mon810 real-time PCR assays were developed in which the event specificity was based on the detection of transgene and plant rearranged sequences found to 3' flank the insertion site. Every assay was specifically designed for one particular detection chemistry, that is, AmpliFluor, Molecular Beacon, MGB, TaqMan, and SYBR-Green I. When possible, the assays were adapted to fast cycling mode. All assays displayed satisfactory performance parameters, although Molecular Beacon, MGB, and TaqMan chemistries were the most suitable for quantification purposes in both conventional and fast cycling modes.     

Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods

The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.     

Development of a real-time PCR for the detection of lupine DNA (lupinus species) in foods

Lupine flour, protein, and fiber have become common ingredients in food products. The association of lupine-related allergic incidents with peanut allergy is a cause for concern as the latter may bring about severe reactions. In this study, a hybridization probe-based real-time PCR assay for the detection of lupine DNA in foods was developed. Particular attention was paid to the specificity of the method, which was verified by analysis of DNA extracts from more than 50 potential food ingredients such as legumes, cereals, seeds, nuts, spices, fruits, and meat. The limit of detection of the method was determined as 0.1 mg/kg. The successful detection of the presence/absence of lupine DNA in 20 samples proved the suitability of the assay for the analysis of frequently encountered food matrices.     

Assessing effects of earthworm cast on methanotrophic community in a soil biocover by concurrent use of microarray and quantitative real-time PCR

Effects of earthworm cast (EC) on the methanotrophic community in a soil biocover were evaluated using microarray and quantitative real-time PCR (qRT-PCR). Soil was collected from a biocover with either soil alone or a mixture of soil and EC (3:1, w/w). The microarray results showed a more diverse methanotrophic community in the EC biocover than that in the soil biocover (p < 0.05). A principal component analysis result confirmed a substantial change in the methanotrophic community structure due to the added EC. Type I methanotrophs dominated both biocovers, with Methylobacter being most abundant. The qRT-PCR results showed that EC greatly increased the methanotrophic population levels (p < 0.05) up to 100 fold. In conclusion, EC can increase the population density of methanotrophs as well as their diversity, resulting in a substantial shift in the community structure. The results confirmed the promising potential of EC as a bed material for enhancing the biocover performance.     

Real-time quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods

A fast and quantitative method was developed to detect transgenic "Maximizer" maize "event 176" (Novartis) and "Roundup Ready" soybean (Monsanto) in food by real-time quantitative PCR. The use of the ABI Prism 7700 sequence detection system allowed the determination of the amplified product accumulation through a fluorogenic probe (TaqMan). Fluorescent dyes were chosen in such a way as to coamplify total and transgenic DNA in the same tube. Using real-time quantitative PCR, 2 pg of transgenic or total DNA per gram of starting sample was detected in 3 h after DNA extraction and the relative amounts of "Maximizer" maize and "Roundup Ready" soybean in some representative food products were quantified.     

Comparison of DNA extraction methods and development of duplex PCR and real-time PCR to detect tomato, carrot, and celery in food

Traceability is of particular importance for those persons who suffer allergy or intolerance to some food component(s) and need a strict avoidance of the allergenic food. In this paper, methodologies are described to fingerprint the presence of allergenic species such as carrot, tomato, and celery by DNA detection. Three DNA extraction methods were applied on vegetables and foods containing or not containing the allergens, and the results were compared and discussed. Fast SYBR Green DNA melting curve temperature analyses and duplex PCR assays with internal control have been developed for detection of these allergenic vegetables and have been tested on commercial foods. Spiking food experiments were also performed, assessing that limits of detection (LOD) of 1 mg/kg for carrot and tomato DNA and 10 mg/kg for celery DNA have been reached.     

Development of a real-time PCR and a sandwich ELISA for detection of potentially allergenic trace amounts of peanut (Arachis hypogaea) in processed foods

Hidden allergens in food products are, especially for peanut-allergic consumers, a serious problem because even low amounts (approximately 200 microg) of peanut can elicit allergic reactions. Undeclared peanut traces can be found in processed food products, because contaminations with peanut during production processes are frequent. To minimize the risk of such cross-contaminations, it is necessary to develop sensitive analytical methods for the detection of hidden allergens in foods. For this approach we developed two peanut-specific assays based on the detection of peanut protein by specific antibodies (sandwich ELISA) and by the detection of peanut-specific DNA (part of the coding region of Ara h 2) by a real-time PCR. Both tests did not show any cross-reactivity with 22 common food ingredients (cereals, nuts, legumes), and the limit of detection is <10 ppm peanut in processed foods. Thirty-three random samples of food products were tested for the presence of peanut to compare both assay types with each other and to evaluate the percentage of foods on the German market that are contaminated with peanut traces. We found that four products (13.3%) without peanut in the list of ingredients contained peanut protein in a range from 1 to 74 ppm peanut protein and that the results of both tests correlated well. The real-time PCR was able to detect one more positive sample than the sandwich ELISA. In conclusion, both assays are sensitive and specific tools for the detection of hidden allergens in processed foods.     

Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments ( approximately 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.     

Disappearance of azoxystrobin, pyrimethanil, cyprodinil, and fludioxonil on tomatoes in a greenhouse

The disappearance of azoxystrobin, pyrimethanil, cyprodinil, and fludioxonil on tomatoes in greenhouse was studied. At the preharvest interval, except for cyprodinil, the pesticide residues were below the MRL fixed in Italy. The mechanism of disappearance studied with model systems shows that the decrease in residues was due to codistillation and photodegradation in pyrimethanil, to photodegradation in fludioxonil, and to evaporation and codistillation in cyprodinil. Azoxystrobin residues were stable during all experiments.