Quantitative detection of the 35S promoter and the NOS terminator using quantitative competitive PCR

 DNA-based analytical methods are often used to verify the presence of genetically modified organisms (GMOs) in food. In Switzerland, a preliminary study, organized by a subcommission of the Swiss Food Manual, of different polymerase chain reaction (PCR) systems for the detection of GMOs showed that the application of qualitative PCR systems can lead to interlaboratory differences of at least a factor of 10. These differences can be diminished using internal standards (competitors). The quantitative competitive (QC) PCR for the detection of the 35S promoter or the NOS terminator in food samples is presented. The GMO content of food samples can be determined using QC-PCR. Received: 2 July 1998 / Revised version: 15 October 1998     

Monitoring of genetically modified food and feed in the Tunisian market using qualitative and quantitative real-time PCR

Genetically modified organisms (GMO) invade more and more the agricultural production in the world. Although there are no legislations on GM labeling and cultivation of GM crops in Tunisia, the present study aims to check the status of GMO in Tunisian market using qualitative and quantitative real time-PCR (QRT-PCR). Three-hundred-sixty five samples were collected and different DNA extraction methods were adapted and optimized. Specific primers targeting 35S promoter from Cauliflower mosaic virus (CaMV) and nopaline synthase terminator from Agrobacterium tumefaciens (At) were used for the detection of the GMO insert and Taxon specific primers for the detection of plant species. Validated Taqman® probes (EU-RL) targeting event specific regions of the maize events MON810, Bt11, and the soybean event RRS were used for the quantification studies. Seven food and feed products showed different amounts of RRS (1.9%), MON810 (2.1%), and Bt11 (1.6%). The results demonstrate for the first time the presence of GMO in Tunisian markets reinforcing the need for the development of accurate quantitative methods in routine analyses.     

Detection of eight GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU and several other countries requires that foods containing genetically modified organisms (GMOs) should be approved and labelled. This has necessitated the development of methods for detection of such materials. For screening purposes these methods should preferably enable detection of several different GMOs. Here we present a simple, robust, qualitative, nineplex PCR method for event-specific detection of maize T25, GA21, TC1507, MON863, MON810, NK603, construct specific detection of BT176, BT11 and detection of the endogenous hmga maize reference gene. PCR is carried out with primers labelled with fluorescent groups and the amplicons are detected using fluorescence capillary electrophoresis. Using mixtures of DNA from different certified reference materials, the detection limit was determined to approximately 0.1% for each GMO. Good agreement was observed in 85 of 88 determinations when eleven food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5′-nuclease PCR. Discrepancies were only observed for one GMO at or close to the detection limit. The presented method is therefore suitable for screening purposes for food and feed containing the most common maize GMOs.     

A quantitative competitive PCR system to detect contamination of wheat, barley or rye in gluten-free food for coeliac patients

 The only treatment in coeliac disease is a lifelong avoidance of wheat, barley and rye (WBR) in the daily nutrition. According to the Codex Alimentarius Commission of the Joint Food and Agricultural Organisation and the World Health Organisation of the United Nations, 100 ppm gliadin (10 mg gliadin/100 g dry weight) is the maximum allowed in food labelled as 'gluten-free'. The present study describes the evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) system as an indication of contamination of gluten-free food with the coeliac-toxic cereals. The QC-PCR system simultaneously detects WBR-DNA on the basis of a non-coding region of chloroplast trnL gene. An internal DNA standard was constructed by adding 20-bp to the original PCR product. This standard was calibrated to 0.02% and 0.2% wheat DNA corresponding to 10 ppm and 100 ppm gliadin, respectively. The QC-PCR system was applied to 15 commercially available products labelled as 'gluten-free'. QC-PCR and ELISA yielded identical results for most cases. By QC-PCR as well as by ELISA one sample was shown to contain more than the allowed maximum limit to be labelled as 'gluten-free'. Through the application of both methods, the origin of a contamination can be identified. A positive QC-PCR signal and a negative ELISA result indicates a possible gliadin-free wheat starch addition and vice versa a possible addition of wheat-free gliadin as a food additive. Both methods support each other in testing gluten-free products. Received: 23 March 2000     

Parallelised real-time PCR for identification of maize GMO events

Identification of specific material derived from genetically modified organisms (GMO) present in food, feed or seed samples screened positive for the presence of genetic modification(s) is mandatory for the official food and feed control in the European Union. Since the introduction of regulation (EC) No 1829/2003 in 2004, the number of maize GMO events either approved in the EU or with a pending application grew constantly. By the sheer multitude of events and crossed events (stacks), maize poses a special challenge on official food and feed control. We developed a modular qualitative detection system for the parallel identification of maize GMO events to cope with the increasing number of GMO potentially present in routine samples. This system is based on validated real-time PCR assays in a microtitre plate format grouped modularly by crop species. The maize module identifies in parallel, i.e. simultaneously, 15 maize events and RoundupReady soy in a single analytical run of approximately 2 h. Maize modules can be conveniently prepared in advance and stored at −20 °C until use. Ready-to-use reference DNA mixtures serve as positive controls. The modular approach is flexible as it allows easy change or addition of individual detection reactions, if necessary, e.g. when new validated methods become available. 23 food, 14 feed and 8 seed samples were successfully analysed with the maize module. The parallel detection of nine different GMO maize and soy events in single routine samples demonstrated the usefulness of the parallelised modular approach for routine GMO analysis.     

Collaborative Study of a T-nos Real-Time PCR Method for Screening of Genetically Modified Organisms in Food Products

In the EU the presence of genetically modified organisms (GMO) in food is controlled by the Member States official laboratories within their national inspection and monitoring programs. The most frequently used approach to detect the presence of GMO material in different food matrices is the PCR-based screening for the CaMV 35S promoter and the nos terminator (T-nos) DNA sequence from Agrobacterium tumefaciens. A real-time PCR method for detection of T-nos and its validation in a collaborative trial study is described in this paper. The PCR system amplifies a 84 bp fragment and showed to be sensitive to detect at least 5 copies of the T-nos DNA sequence. The assay was adapted for use in real-time PCR instruments using plastic reaction vials and glass capillaries, respectively. A total of 24 laboratories participated in the study and each laboratory received 18 DNA blind samples prepared from GMO certified reference materials (0.1 % and 0.5 % NK601 maize) and from a non-GM maize flour. All samples containing NK603 maize DNA were correctly classified as T-nos positive by the participating laboratories. For the blank samples (0 % GMO) only three false-positive results were reported. A detailed evaluation of the results of the collaborative trial study is described. Received: March 19, 2007     

Collaborative Study of a T-nos Real-Time PCR Method for Screening of Genetically Modified Organisms in Food Products

In the EU the presence of genetically modified organisms (GMO) in food is controlled by the Member States official laboratories within their national inspection and monitoring programs. The most frequently used approach to detect the presence of GMO material in different food matrices is the PCR-based screening for the CaMV 35S promoter and the nos terminator (T-nos) DNA sequence from Agrobacterium tumefaciens. A real-time PCR method for detection of T-nos and its validation in a collaborative trial study is described in this paper. The PCR system amplifies a 84 bp fragment and showed to be sensitive to detect at least 5 copies of the T-nos DNA sequence. The assay was adapted for use in real-time PCR instruments using plastic reaction vials and glass capillaries, respectively. A total of 24 laboratories participated in the study and each laboratory received 18 DNA blind samples prepared from GMO certified reference materials (0.1 % and 0.5 % NK601 maize) and from a non-GM maize flour. All samples containing NK603 maize DNA were correctly classified as T-nos positive by the participating laboratories. For the blank samples (0 % GMO) only three false-positive results were reported. A detailed evaluation of the results of the collaborative trial study is described.     

Quantitative competitive (QC) PCR for quantification of porcine DNA

Many meat products nowadays may contain several species in different proportions. To protect consumers from fraud and misdeclarations, not only a qualitative but also a quantitative monitoring of ingredients of complex food products is necessary. DNA based techniques like the polymerase chain reaction (PCR) are widely used for identification of species but no answer to the proportional amount of a certain species could be given using current techniques. In this study we report the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of porcine DNA using a new porcine specific PCR system based on the growth hormone gene of sus scrofa. A DNA competitor differing by 30 bp in length from the porcine target sequence was constructed and used for PCR together with the target DNA. Specificity of the new primers was evaluated with DNA from cattle, sheep, chicken and turkey. The competitor concentration was adjusted to porcine DNA contents of 2 or 20% by coamplification of mixtures containing porcine and corresponding amounts of bovine DNA in defined ratios.     

A PCR-microarray method for the screening of genetically modified organisms

A new method to screen and to identify genetically modified organisms (GMO) is presented in this paper. It is based on the detection of multiple genetic elements common to GMO by their amplification via PCR followed by direct hybridisation of the amplicons on microarray. The pattern of the elements is then compared to a database of the composition of EU-approved GMO and an identification of the GMO is then proposed. The limit of detection of the method was ≤0.1% GMO content (w/w) expressed as the amount of target DNA present in the template for single unprocessed material. The DNA targets were detected both in reference materials and in mixtures with the same detection limit. The specificity for the detection of the different elements was found to be very good with no cross-reaction even in samples with two GMO present at different concentrations. The paper presents examples of GMO identification and discusses the potential and limitation of such approaches and how they can facilitate the work of private and enforcement detection laboratories.     

Detection of DNA in soybean oil

 The presence of DNA in foodstuffs which are, or contain, genetically modified organisms (GMO) is the basic requirement for labelling of GMO food in Switzerland and is also being discussed as a requirement for labelling in the European Union. The present work presents data indicating that no genetic material can be recovered after the first processing steps of soybean oil, i.e. when crude soybean oil is simply centrifuged. This fact is of some relevance because centrifugation is one of the first steps in industrial oil processing. A nested PCR system utilising a single copy gene (lectin 1) showed that centrifugation purified the oil from the genetic material by at least a factor of 10 000. The same results were observed when industrially processed oil fractions were analysed. Thus, with respect to the presence of DNA, soybean oil from GMO soybeans is identical to traditional oil and does not need to be labelled as a GMO product in Switzerland. Received: 21 January 1998 / Revised version: 30 March 1998     

Detection of DNA in soybean oil

 The presence of DNA in foodstuffs which are, or contain, genetically modified organisms (GMO) is the basic requirement for labelling of GMO food in Switzerland and is also being discussed as a requirement for labelling in the European Union. The present work presents data indicating that no genetic material can be recovered after the first processing steps of soybean oil, i.e. when crude soybean oil is simply centrifuged. This fact is of some relevance because centrifugation is one of the first steps in industrial oil processing. A nested PCR system utilising a single copy gene (lectin 1) showed that centrifugation purified the oil from the genetic material by at least a factor of 10 000. The same results were observed when industrially processed oil fractions were analysed. Thus, with respect to the presence of DNA, soybean oil from GMO soybeans is identical to traditional oil and does not need to be labelled as a GMO product in Switzerland. Received: 21 January 1998 / Revised version: 30 March 1998     

Quantitative,multiplex ligation-dependent probe amplification for the determination of eight genetically modified maize events

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. To this end, we have developed a robust, quantitative, sensitive, nine-plex ligation-dependent probe amplification method, GMO-MLPA, for event-specific detection of maize TC1507, MON810, NK603, MON863, BT176, T25, GA21, construct-specific detection of BT11, and detection of the endogenous hmga maize reference gene. Ligated probes are amplified by PCR. Amplicons are detected using capillary electrophoresis. Specific GMO signals are normalised relative to the signal from the endogenous hmga gene and quantified by comparing with known standard curves. The method is suitable for quantification in the 0–2% range. Agreement was obtained in 149 of 160 determinations when 11 known mixtures of GMO and 9 food and feed samples were analysed using the GMO-MLPA method and compared to results from quantitative real-time 5′-nuclease PCR. The presented method is, therefore, suitable for quantification purposes for food and feed containing the most common maize GMOs.     

Detection of genetically modified soy in processed foods sold commercially in Malaysia by PCR-based method

Regulations for the use and labeling of genetically modified organism (GMO) products and derived ingredients are being implemented worldwide, that demands reliable and accurate methods to detect GMO in raw materials and food products. In this study, polymerase chain reaction (PCR) method was established for monitoring products derived from GMO that are sold in the markets in Malaysia, which specifically amplify the 35S promoter, nos (nopaline synthase-terminator), EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) and RRS (CTP/CP4EPSPS). Using this method, we investigated the incidence of genetically modified soy (GM-soy) and specifically the presence of roundup ready soy (RRS). All the soybean samples were evidenced by presence of the lectin gene. Out of 85 samples examined, the 18 positive GM samples were raw bean (9), tofu (8) and tempe (1) (a traditional Malay food). The results demonstrate for the first time the presence of GM-soy in Malaysian food products, reinforcing the need for the development of accurate quantitative methods for routine analyses.     

Validation of the performance of a GMO multiplex screening assay based on microarray detection

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip^® GMO, Eppendorf, Germany). The validation was performed within the framework of a European project (Co-Extra, contract no 007158) and in collaboration with 12 laboratories specialised in GMO detection. The present study reports the strategy and the results of an ISO complying validation of the method carried out through an inter-laboratory study. Sets of blind samples were provided consisting of DNA reference materials covering all the elements detectable by specific probes present on the array. The GMO concentrations varied from 1% down to 0.045%. In addition, a mixture of two GMO events (0.1% RRS diluted in 100% TOPAS19/2) was incorporated in the study to test the robustness of the assay in extreme conditions. Data were processed according to ISO 5725 standard. The method was evaluated with predefined performance criteria with respect to the EC CRL method acceptance criteria. The overall method performance met the acceptance criteria; in particular, the results showed that the method is suitable for the detection of the different target elements at 0.1% concentration of GMO with a 95% accuracy rate. This collaborative trial showed that the method can be considered as fit for the purpose of screening with respect to its intra- and inter-laboratory accuracy. The results demonstrated the validity of combining multiplex PCR with array detection as provided by the DualChip^® GMO (Eppendorf, Germany) for the screening of GMO. The results showed that the technology is robust, practical and suitable as a screening tool.     

Development and applications of real-time PCR standards for GMO quantification based on tandem-marker plasmids

The need to comply with mandatory thresholds introduced for the labeling of GM food and feed by recent European Regulations is increasing the interest for reliable standards for GMO quantification. Certified reference materials (CRMs) for the different GMO lines are an essential element to calibrate measurement systems adopted to quantify GM ingredients. Unfortunately, conventional CRMs are affected not only by their high production costs but also by time consuming production and certification procedures. At the moment CRMs are available only for few of the GMOs authorized in Europe. In addition, latest developments in European legislation makes conceivable that the number of different authorized GM lines to be analysed will increase significantly, raising the need for additional CRMs. In the field of GMO detection and quantification, plasmids have been demonstrated to represent a cheap and reliable alternative to conventional CRMs. Here we describe the production of tandem-marker plasmids containing simultaneously specific target sequences for the GMO of interest and a species-specific reference marker sequence in a 1:1 ratio have been developed and used as real-time PCR standards. Strengths and weaknesses of these novel standards are discussed in detail.E. Mattarucchi and F. Weighardt: first authors     

Quantitative detection method for Roundup Ready soybean in food using duplex real‐time PCR MGB chemistry

BACKGROUND: Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non‐authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost‐efficient solution. RESULTS: Construct‐specific primers and probe were developed for quantitative analysis of Roundup Ready ^® soybean (RRS) event glyphosate‐tolerant soybean (GTS) 40‐3‐2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS‐ and Le1‐specific quantitative real‐time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. CONCLUSION: In this study, a duplex qRTPCR using TaqMan minor groove binder‐non‐fluorescent quencher (MGB‐NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40‐3‐2 that can be used for practical monitoring in processed food products. Copyright © 2010 Society of Chemical Industry     

Applicability of the “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” in processed maize matrices

Simple tools to detect the presence of genetically modified organisms (GMO) in commercial products represent a valuable aid in managing the legal requirements for GMO testing in a cost-effective way. The “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” was developed to meet such requirements and was here further tested for its applicability on detecting GMO in recalcitrant maize matrices. Sixty-four processed maize products were purchased from the market of the European Union and analyzed for their GMO content. Seventy-five percent of the test samples were positive for the presence of GMO. In two samples, trace amounts of the so-called asynchronously authorized GMO could be detected. The overall outcome of the analyses indicated that most of the commercially available genetically modified maize events for food use could be detected in this study. Finally, the possibility to use the “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” in detecting GMO at a 0.1% mass level is documented. The implications of these results on the further development of such type of PCR-based GMO detection systems are discussed.     

Real-time PCR multiplex method for the quantification of Roundup Ready soybean in raw material and processed food

This paper describes a quantitative real-time multiplex PCR method optimised for the ABI PRISM^® 7700 Sequence Detection System (SDS) and TaqMan^® chemistry for Roundup Ready^® Soybean (RRS) in raw material and processed food. This method has the advantage of performing the amplification of the target taxon lectin gene and the genetically modified (GM) target sequence in the same test tube. The quantification is based on a calibration curve obtained with the DNA extracted from Certified Reference Material standards (CRM IRMM-410: R) in the range 0.1–5% RRS. The method was validated in-house by using CRMs and the applicability was verified on three mixtures of soybean flour at 1, 5, 10% of RRS respectively and on prepared baked products (biscuits and plum cakes). The statistical parameters of the method were found to be satisfactory both for soybean flour and baked products. In the process the absolute LOD and LOQ was 7.1 and 35.5 copy number respectively; the relative LOD and LOQ was 0.03 and 0.01% of RRS respectively.