Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction

Transgenic soybean line GTS-40-3-2, marketed under the trade name Roundup Ready (RR) soy, was developed by Monsanto (USA) to allow for the use of glyphosate, the active ingredient of the herbicide Roundup, as a weed control agent. RR soy was first approved in Canada for environmental release and for feed products in 1995 and later for food products in 1996 and is widely grown in Canada. Consumer concern issues have resulted in proposed labeling regulations in Canada for foods derived from genetically engineered crops. One requirement for labeling is the ability to detect and accurately quantify the amount of transgenic material present in foods. Two assays were evaluated. A conventional qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of soy and RR soy and a real-time PCR to quantify the amount of RR soy present in samples that tested positive in the first assay. PCR controls consisted of certified RR soy reference material, single transgenic soybeans, and a processed food sample containing a known amount of RR soy. To test real-world applicability, a number of common grocery store food items that contain soy-based products were tested. For some samples, significant differences in amplification efficiencies during the quantitative PCR assays were observed compared to the controls, resulting in potentially large errors in quantification. A correction factor was used to try to compensate for these differences.     

Quantitation of Bt-176 maize genomic sequences by surface plasmon resonance-based biospecific interaction analysis of multiplex polymerase chain reaction (PCR)

Surface plasmon resonance (SPR) based biosensors have been described for the identification of genetically modified organisms (GMO) by biospecific interaction analysis (BIA). This paper describes the design and testing of an SPR-based BIA protocol for quantitative determinations of GMOs. Biotinylated multiplex Polymerase Chain Reaction (PCR) products from nontransgenic maize as well as maize powders containing 0.5 and 2% genetically modified Bt-176 sequences were immobilized on different flow cells of a sensor chip. After immobilization, different oligonucleotide probes recognizing maize zein and Bt-176 sequences were injected. The results obtained were compared with Southern blot analysis and with quantitative real-time PCR assays. It was demonstrated that sequential injections of Bt-176 and zein probes to sensor chip flow cells containing multiplex PCR products allow discrimination between PCR performed using maize genomic DNA containing 0.5% Bt-176 sequences and that performed using maize genomic DNA containing 2% Bt-176 sequences. The efficiency of SPR-based BIA in discriminating material containing different amounts of Bt-176 maize is comparable to real-time quantitative PCR and much more reliable than Southern blotting, which in the past has been used for semiquantitative purposes. Furthermore, the approach allows the BIA assay to be repeated several times on the same multiplex PCR product immobilized on the sensor chip, after washing and regeneration of the flow cell. Finally, it is emphasized that the presented strategy to quantify GMOs could be proposed for all of the SPR-based, commercially available biosensors. Some of these optical SPR-based biosensors use, instead of flow-based sensor chips, stirred microcuvettes, reducing the costs of the experimentation.     

Development of sensitive crop-specific polymerase chain reaction assays using 5S DNA: applications in food traceability

The 5S intergenic spacers were amplified using a common pair of primers and sequenced from four species (Brassica napus, Zea mays, Helianthus annuus, and Glycine max). Crop-specific assays were developed from primers designed from the spacers and tested to amplify corresponding DNAs in both conventional end-point and real-time polymerase chain reactions (PCRs). The high copy numbers of the 5S DNA in plants make it possible to detect very small amounts of DNA using this marker. This sensitivity made it possible to compare different DNA extraction methods for highly processed food products using 5S spacers, even allowing dilution of templates to overcome PCR inhibition.     

Detection of ruminant meat and bone meals in animal feed by real-time polymerase chain reaction: result of an interlaboratory study

The commercialization of animal feeds infected by prions proved to be the main cause of transmission of bovine spongiform encephalopathy (BSE). Therefore, feed bans were enforced, initially for ruminant feeds, and later for all feeds for farmed animals. The development and validation of analytical methods for the species-specific detection of animal proteins in animal feed has been indicated in the TSE (Transmissible Spongiform Encephalopathies) Roadmap (European Commission. The TSE (Transmissible Spongiform Encephalopathy) roadmap. URL: http://europa.eu.int/comm/food/food/biosafety/bse/roadmap_en.pdf, 2005) as the main condition for lifting the extended feed ban. Methods based on polymerase chain reaction (PCR) seem to be a promising solution for this aim. The main objective of this study was to determine the applicability of four different real-time PCR methods, developed by three National expert laboratories from the European Union (EU), for the detection and identification of cattle or ruminant species in typical compound feeds, fortified with meat and bone meals (MBM) from different animal species at different concentration levels. The MBM samples utilized in this study have been treated using the sterilization condition mandatory within the European Union (steam pressure sterilization at 133 degrees C, 3 bar, and 20 min), which is an additional challenge to the PCR methods evaluated in this study. The results indicate that the three labs applying their PCR methods were able to detect 0.1% of cattle MBM, either alone or in mixtures with different materials such as fishmeal, which demonstrates the improvement made by this technique, especially when compared with results from former interlaboratory studies.     

Molecular analysis of the DNA polymorphism of wild barley (Hordeum spontaneum) germplasm using the polymerase chain reaction

Summary Hordeum spontaneum is the progenitor of cultivated barley (H. vulgare) and is an important source of genetic variation for barley breeding programs. The genetic diversity ofH. spontaneum in the Australian germplasm collection was investigated using the polymerase chain reaction with random and semi-random primers. This approach was found to be robust in respect of reaction conditions. Genetic dissimilarity values between genotypes were used to produce a phenogram of the relationships among the accessions using the unweighted pair group method with arithmetic mean. The largest divergence was observed among Israeli accessions, whereas the Turkish and Iranian samples clustered as distinct subsets, each apparently related to portion of the Israeli material. The results indicate that the genetic diversity of the wild barleys is broadly correlated with geographic distribution.     

Detection of genetically modified maize MON810 and NK603 by multiplex and real-time polymerase chain reaction methods

In this study, the event-specific primers for insecticide-resistant maize, MON810, and herbicide-tolerance maize, NK603, have been designed. Simplex PCR and multiplex PCR detection method have been developed. The detection limit of the multiplex PCR is 0.5% for MON810 and NK603 in 50 ng of the template for one reaction. Quantitative methods based on real-time quantitative PCR were developed for MON810 and NK603. Plasmid pMulM2 as reference molecules for the detection of MON810 and NK603 was constructed. Quantification range was from 0.5 to 100% in 100 ng of the DNA template for one reaction. The precision of real-time Q-PCR detection methods, expressed as coefficient of variation for MON810 and NK603 varied from 1.97 to 8.01% and from 3.45 to 10.94%, respectively. The range agreed with European interlaboratories test results (25%). According to the results, the methods for quantitative detection of genetically modified maize were acceptable.     

Real-time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on commercial samples

Mozzarella cheese obtained from buffalo (Bubalus bubalis) milk is a typical Italian product certificated by means of the European Protected Designation of Origin (PDO). Mozzarella cheese can also be obtained from bovine milk or bovine/buffalo milk mixtures, but in this case, it cannot be sold as PDO product, and its label must report the actual ingredients. However, bovine milk in PDO products was frequently detected in the past, suggesting fraudulent addition or accidental contamination. Several methods based on end-point polymerase chain reaction (PCR) have been profitably applied in a large number of tests to detect the presence of undeclared ingredients, also in dairy products. In the present study we report a real-time PCR method able to quantify bovine milk addition to pure buffalo cheese products. We validated a normalized procedure based on two targets: bovine mitochondrial cytochrome b (cyt b) to detect and quantify the bovine DNA and nuclear growth hormone (GH) gene used as a universal reference marker. With the use of this real-time PCR assay, 64 commercial mozzarella di bufala cheese samples purchased at local supermarkets, dairy shops, or directly from cheese manufacturers were analyzed. The results obtained demonstrate that most of the commercial samples were contaminated with bovine milk. Therefore, this assay could be conveniently employed to carry out routine and accurate controls aimed not only to discourage any fraudulent behavior but also to reduce risks for consumer health.     

Qualitative and quantitative polymerase chain reaction analysis for genetically modified maize MON863

Qualitative and quantitative analytical methods were developed for the new event of genetically modified (GM) maize, MON863. One specific primer pair was designed for the qualitative polymerase chain reaction (PCR) method. The specificity and sensitivity of the designed primers were confirmed. PCR was performed on genomic DNAs extracted from MON863, other GM events, and cereal crops. Single PCR product was obtained from MON863 by the designed primer pair. Eight test samples including GM maize MON863 were prepared at 0.01 approximately 10% levels and analyzed by PCR. Limit of detection of the method was 0.01% for GM maize MON863. On the other hand, another specific primer pair and probe were also designed for quantitative method using a real-time polymerase chain reaction. As a reference molecule, a plasmid was constructed from a taxon-specific DNA sequence for maize, a universal sequence for a cauliflower mosaic virus (CaMV) 35S promoter used in most genetically modified organisms, and a construct-specific DNA sequence for the MON863 event. Six test samples of 0.1, 0.5, 1.0, 3.0, 5.0 and 10.0% of GM maize MON863 were quantitated for the validation of this method. At the 3.0% level, the bias (mean vs true value) for MON863 was 3.0%, and its relative standard deviation was 5.5%. Limit of quantitation of the method was 0.5%. These results show that the developed PCR methods can be used to qualitatively and quantitatively detect GM maize MON863.     

Interlaboratory evaluation of a real-time multiplex polymerase chain reaction method for identification of salmon and trout species in commercial products

This interlaboratory study evaluated a real-time multiplex polymerase chain reaction (PCR) method for identification of salmon and trout species in a range of commercial products in North America. Eighty salmon and trout products were tested with this method by three independent laboratories. Samples were collected in the United States and Canada, and only the collecting institution was aware of the species declaration. Following analysis with real-time PCR, all three laboratories were able to identify species in 79 of the 80 products, with 100% agreement on species assignment. A low level of fraud was detected, with only four products (5%) found to be substituted or mixtures of two species. The results for two of the fraudulent products were confirmed with alternate methods, but the other two products were heavily processed and could not be verified with methods other than real-time PCR. Overall, the results of this study show the usefulness and versatility of this real-time PCR method for the identification of commercial salmon and trout species.     

Development and comparison of four real-time polymerase chain reaction systems for specific detection and quantification of Zea mays L

Four real-time polymerase chain reaction systems aiming at the specific detection and quantification of maize DNA are described. They have been developed in four independent laboratories targeting different maize sequences, i.e., alcohol dehydrogenase (Adh1), high mobility group protein (hmga), invertase A (ivr1), and zein, respectively. They were all fully specific, showing a very similar quantification accuracy along a number of distantly related maize cultivars and being either single or low copy number genes. They were highly sensitive and exhibited limits of quantification below 100 maize genomic copies. In consequence, they are considered suitable for use as maize specific endogenous reference genes in DNA analyses, including GMO quantitative tests.     

Identification of gadoid species in fish meat by polymerase chain reaction (PCR) on genomic DNA

Identification of fish species is significant due to the increasing interest of consumers in the meat of sea fish. Methods focusing on fish species identification help to reveal fraudulent substitution among economically important gadoid species in commercial seafood products. The objective of this work was to develop a conventional PCR method for the differentiation of the following gadoid fish species in fish products: Alaska pollack ( Theragra chalcogramma), blue whiting ( Micromesistius poutassou), hake spp. ( Merluccius spp.), Atlantic cod ( Gadus morhua), saithe ( Pollachius virens), and whiting ( Merlangius merlangus). The species-specific primer pairs for gadoid species determination were based on the partial pantophysin I ( PanI) genomic sequence. Sequence identification was confirmed by cloning and sequencing of the PCR products obtained from the species considered. For the simultaneous detection of Alaska pollack, blue whiting, and hake spp., a quadruplex PCR system was constructed. Other gadoid species were detected in separate PCR reactions. After optimization of the reactions, the developed PCR systems were used for the analysis of codfish samples obtained from the Czech market and the customs' laboratories. This method represents an alternative approach in the use of genomic DNA for the identification of fish species. This method is rapid, simple, and reliable without the need for further confirmative methods. Furthermore, the identification of a mixture of more than one species is possible. The PCR system has been optimized for routine diagnostic purposes.     

Event-specific qualitative and quantitative polymerase chain reaction analysis for genetically modified canola T45

Polymerase chain reaction (PCR) methods have been the main technical support for the detection of genetically modified organisms (GMOs). To date, GMO-specific PCR detection strategies have been developed basically at four different levels, such as screening-, gene-, construct-, and event-specific detection methods. Event-specific PCR detection method is the primary trend in GMO detection because of its high specificity based on the flanking sequence of exogenous integrant. GM canola, event T45, with tolerance to glufosinate ammonium is one of the commercial genetically modified (GM) canola events approved in China. In this study, the 5'-integration junction sequence between host plant DNA and the integrated gene construct of T45 canola was cloned and revealed by means of TAIL-PCR. Specific PCR primers and TaqMan probes were designed based upon the revealed sequence, and qualitative and quantitative TaqMan real-time PCR detection assays employing these primers and probe were developed. In qualitative PCR, the limit of detection (LOD) was 0.1% for T45 canola in 100 ng of genomic DNA. The quantitative PCR assay showed limits of detection and quantification (LOD and LOQ) of 5 and 50 haploid genome copies, respectively. In addition, three mixed canola samples with known GM contents were detected employing the developed real-time PCR assay, and expected results were obtained. These results indicated that the developed event-specific PCR methods can be used for identification and quantification of T45 canola and its derivates.     

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.     

Real-time polymerase chain reaction monitoring of recombinant DNA entry into soil from decomposing roundup ready leaf biomass

Glyphosate-tolerant, Roundup Ready (RR) soybeans account for about 57% of all genetically modified (GM) crops grown worldwide. The entry of recombinant DNA into soil from GM crops has been identified as an environmental concern due to the possibility of their horizontal transfer to soil microorganisms. RR soybeans contain recombinant gene sequences that can be differentiated from wild-type plant and microbial genes in soil by using a sequence-specific molecular beacon and real-time polymerase chain reaction (PCR). A molecular beacon-based real-time PCR system to quantify a wild-type soybean lectin ( le1) gene was designed to compare amounts of endogenous soybean genes to recombinant DNA in soil. Microcosm studies were carried out to develop methodologies for the detection of recombinant DNA from RR soybeans in soil. RR soybean leaf litterbags were imbedded in the soil under controlled environmental conditions (60% water holding capacity, 10/15 degrees C, and 8/16 h day/night) for 30 days. The soybean biomass decomposition was described using a single-phase exponential equation, and the DNA concentration in planta and in soil was quantified using real-time PCR using sequence-specific molecular beacons for the recombinant cp4 epsps and endogenous soybean lectin ( le1) genes. The biomass of RR soybean leaves was 8.6% less than nontransgenic (NT) soybean leaves after 30 days. The pooled half-disappearance time for cp4 epsps and le1 in RR and of le1 in NT soybean leaves was 1.4 days. All genes from leaves were detected in soil after 30 days. This study provides a methodology for monitoring the entry of RR and NT soybean DNA into soil from decomposing plant residues.     

Application of relative quantification TaqMan real-time polymerase chain reaction technology for the identification and quantification of Thunnus alalunga and Thunnus albacares

A novel one-step methodology based on real-time Polymerase Chain Reaction (PCR) technology has been developed for the identification of two of the most valuable tuna species. Nowadays, species identification of seafood products has a major concern due to the importing to Europe of new species from other countries. To achieve this aim, two specific TaqMan systems were devised to identify Thunnus alalunga and Thunnus albacares. Another system specific to Scombroidei species was devised as a consensus system. In addition, a relative quantification methodology was carried out to quantify T. alalunga and T. albacares in mixtures after the relative amount of the target was compared with the consensus. This relative quantification methodology does not require a known amount of standard, allowing the analysis of many more samples together and saving costs and time. The utilization of real-time PCR does not require sample handling, preventing contamination and resulting in much faster and higher throughput results.     

Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA

An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.     

Use of real-time polymerase chain reaction (PCR) and transformation assay to monitor the persistence and bioavailability of transgenic genes released from genetically modified papaya expressing nptII and PRSV genes in the soil

Soil samples were collected from an isolated field from December 2003 to April 2004 where transgenic papaya were planted, and the persistences of transgenic genes of 796 bp (located between 35S promoter and coat protein, 35S-P/PRSV-CP), 398 bp (located between plasmid pBI121 and NOS terminator, pBI121/NOS-T), and 200 bp (located between NOS promoter and nptII gene, NOS-P/nptII) were studied. At the end of planting, the residues of 398 bp in the soil was 0.06 microg g(-1) of soil, whereas the residues of 769 and 200 bp were less than 30 pg g(-1) of soil (detection limit). Kinetics studies on the persistence of these three fragments in sterile distilled water and nonsterile soil microcosms showed that two mechanisms might be involved: an initial fast exponential degradation pattern in the first week and then followed by a slow-release pattern throughout the experiment. Persistence of transgenic DNA in sterile water was longer than in nonsterile soil microcosms, indicating that enzymatic degradation and soil adsorption played important roles on the persistence of DNA in the environment. The reason for the fragment of 398 bp persisted longer than fragments of 769 and 200 bp is not clear, but the guanine plus cytosin (G plus C) content in the DNA fragment might be involved in the stability of DNA in the environment. Biological availability of soil DNA to bacteria conducted by the transformation assay indicated that gene transformation from soil DNA extracts to two Acinetobacter spp. did not occur.     

Quantification of genetically modified soybean by quenching probe polymerase chain reaction

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

Genetically modified organisms in food-screening and specific detection by polymerase chain reaction

PCR methods for the detection of genetically modified organisms (GMOs) were developed that can be used for screening purposes and for specific detection of glyphosate-tolerant soybean and insect-resistant maize in food. Primers were designed to amplify parts of the 35S promoter derived from Cauliflower Mosaic Virus, the NOS terminator derived from Agrobacterium tumefaciens and the antibiotic marker gene NPTII (neomycin-phosphotransferase II), to allow for general screening of foods. PCR/hybridization protocols were established for the detection of glyphosate-tolerant RoundUp Ready soybean and insect-resistant Bt-maize. Besides hybridization, confirmation of the results using restriction analysis was also possible. The described methods enabled a highly sensitive and specific detection of GMOs and thus provide a useful tool for routine analysis of raw and processed food products.     

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.