Increased efficacy for in-house validation of real-time PCR GMO detection methods

To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSD_r and RSD_R) were calculated. The results showed that not only the PCR reaction but also the factors ‘DNA isolation’ and ‘PCR day’ are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods.     

Fuzzy logic-based procedures for GMO analysis

Key to sound validation studies is the formalization and harmonization of procedures for design of experiment and interpretation of results. International guidelines (ISO 5725, ENGL) are available for the validation of GMO detection methods, and ad-hoc validation statistics (e.g. per cent bias, repeatability and reproducibility) are used for in-house and inter-laboratory testing and decision-making. Acceptability criteria have been set but not every situation can be covered by a preset rule; the interpretation of results in validation largely depends on expert judgement being a matter of professional judgment and expertise. Fuzzy logic-based techniques may be used to summarize the information obtained by independent validation statistics and are helpful in such respect. A comprehensive indicator of method performance permits direct comparison between methods and facilitates the evaluation of multiple, yet contradictory statistics. The European Union Reference Laboratory for GM Food and Feed has already proposed the fuzzy principle in the context of method validation. Other studies have also proved its applicability in other areas of GMO analysis, but the application has been limited hitherto. In this article, we review the fuzzy logic principle and its potential to support the continuous progress of GMO science and routine laboratory analyses.     

Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event

In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement must be performed. Moreover, for some reference genes no sufficient information on copy number in and among genomes of different lines is available, making adequate quantification difficult. Once developed, the method was subsequently validated according to IUPAC and ISO 5725 guidelines. Thirteen laboratories from 8 EU countries participated in the trial. Eleven laboratories provided results complying with the predefined study requirements. Repeatability (RSDr) values ranged from 8.7 to 15.9%, with a mean value of 12%. Reproducibility (RSDR) values ranged from 16.3 to 25.5%, with a mean value of 21%. Following Codex Alimentarius Committee guidelines, both the limits of detection and quantitation were determined to be <0.1%.     

The validation of methods for regulatory purposes in the control of residues

The topic of validation is diversified. This review outlines the validation strategies which can be found in national, international and supranational regulations, compares them with one another and aims to elaborate on the main principles. European regulations and legislation, Codex alimentarius guidelines, the official methods program of the AOAC, and naturally the relevant ISO standards, particularly the ISO 5725 series, are taken into consideration. The objective of every validation is to demonstrate fitness for purpose. This varies of course in its characteristics for the diverse uses. However, all approaches have in common the objective of harmonisation of food control by using effective and reliable methods. To this end, criteria are determined and validation models developed and made compulsory. ISO 5725 is the central basis for validations for quantitative methods with its validation specifications through method collaborative studies. On the contrary, there are no valid uniform international method specifications for qualitative methods. Collaborative studies are in opposition to single-lab-validations with different sources of error. Whereas laboratory errors are predominant in collaborative studies, the single-lab-validation or in-house validation concentrates particularly on time and processing errors (intermediate precision). In new statistical models for in-house validations, the matrix mismatch error is also considered. The validation models presented here are of a general nature and can be used in principle for all analytical methods. Correct and appropriate statistical modelling is very important.     

Validation of PCR methods for quantitation of genetically modified plants in food.

For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials.     

Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed

For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LOD_abs) ≤ ten target copies was proven in hexaplex format. A sensitivity ≤ ten target copies of each GMO detection system was still shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel. Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated. The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed samples with high efficiency.     

DNA methods: critical review of innovative approaches

The presence of ingredients derived from genetically modified organisms (GMOs) in food products in the market place is subject to a number of European regulations that stipulate which product consisting of or containing GMO-derived ingredients should be labeled as such. In order to maintain these labeling requirements, a variety of different GMO detection methods have been developed to screen for either the presence of DNA or protein derived from (approved) GM varieties. Recent incidents where unapproved GM varieties entered the European market show that more powerful GMO detection and identification methods will be needed to maintain European labeling requirements in an adequate, efficient, and cost-effective way. This report discusses the current state-of-the-art as well as future developments in GMO detection.     

New trends in bioanalytical tools for the detection of genetically modified organisms: an update

Despite the controversies surrounding genetically modified organisms (GMOs), the production of GM crops is increasing, especially in developing countries. Thanks to new technologies involving genetic engineering and unprecedented access to genomic resources, the next decade will certainly see exponential growth in GMO production. Indeed, EU regulations based on the precautionary principle require any food containing more than 0.9% GM content to be labeled as such. The implementation of these regulations necessitates sampling protocols, the availability of certified reference materials and analytical methodologies that allow the accurate determination of the content of GMOs. In order to qualify for the validation process, a method should fulfil some criteria, defined as “acceptance criteria” by the European Network of GMO Laboratories (ENGL). Several methods have recently been developed for GMO detection and quantitation, mostly based on polymerase chain reaction (PCR) technology. PCR (including its different formats, e.g., double competitive PCR and real-time PCR) remains the technique of choice, thanks to its ability to detect even small amounts of transgenes in raw materials and processed foods. Other approaches relying on DNA detection are based on quartz crystal microbalance piezoelectric biosensors, dry reagent dipstick-type sensors and surface plasmon resonance sensors. The application of visible/near-infrared (vis/NIR) spectroscopy or mass spectrometry combined with chemometrics techniques has also been envisaged as a powerful GMO detection tool. Furthermore, in order to cope with the multiplicity of GMOs released onto the market, the new challenge is the development of routine detection systems for the simultaneous detection of numerous GMOs, including unknown GMOs.     

Advances in molecular techniques for the detection and quantification of genetically modified organisms

Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.     

Evaluation of applied biosystems MicroSeq real-time PCR system for detection of Listeria monocytogenes in food. Performance Tested Method 011002

Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. This report describes the validation of a new Real-Time PCR method to detect Listeria monocytogenes in nine different food matrixes. The complete system consists of the MicroSEQ L. monocytogenes Detection Kit, sample preparation, the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. Two sample preparation methods were validated: the PrepSEQ Nucleic Acid extraction kit and the PrepSEQ Rapid Spin sample preparation kit. The test method was compared to the ISO 11290-1 reference method using an unpaired-study design to detect L. monocytogenes in roast beef, cured bacon, lox (smoked salmon), lettuce, whole cow's milk, dry infant formula, ice cream, salad dressing, and mayonnaise. The MicroSEQ L. monocytogenes Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all food matrixes when the samples were prepared using either of the two sample preparation methods. An independent validation confirmed these findings on smoked salmon and whole cow's milk. The MicroSEQ kit detected all 50 L. monocytogenes strains tested, and none of the 30 nontargeted bacteria strains.     

Screening Methodologies for Genetic Modified Organsims (GMOs)

ABSTRACT

There is an increasing need for analytical methods for the detection of genetically modified organisms (GMOs) detection in food due to the growth of use of GMOs, or their derivatives, in the food industry. This paper aims to briefly introduce the reader to GMOs, to describe the state of the art in detection methods for GMOs, and to provide the reader with the associated terminology, mainly borrowed from molecular biology. The main characteristics of GMO are illustrated and future developments using the biosensor approach for detection are presented.     

Performance Tested Method multiple laboratory validation study of ELISA-based assays for the detection of peanuts in food

Performance Tested Method multiple laboratory validations for the detection of peanut protein in 4 different food matrixes were conducted under the auspices of the AOAC Research Institute. In this blind study, 3 commercially available ELISA test kits were validated: Neogen Veratox for Peanut, R-Biopharm RIDASCREEN FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut. Analyses of the samples were conducted by laboratories representing industry and international and U.S governmental agencies. All 3 commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 microg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be <90%. The probability that a test sample contains an allergen given a prevalence rate of 5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When 2 test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least 2 of the validated kits.     

Evaluation of applied biosystems MicroSEQ real-time PCR system for detection of Salmonella spp. in food

Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time-to-results compared to traditional culture methods. In this study, the MicroSEQ real-time PCR system was evaluated for detection of Salmonella spp. in 10 different food matrixes following the AOAC Research Institute's Performance Tested Method validation program. In addition, the performance of the MicroSEQ system was evaluated for the detection of Salmonella in peanut butter as a part of the Emergency Response Validation Program sponsored by the AOAC Research Institute. The system was compared to the ISO 6579 reference method using a paired-study design for detecting Salmonella spp. in raw ground beef, raw chicken, raw shrimp, Brie cheese, shell eggs, cantaloupe, chocolate, black pepper, dry infant formula, and dry pet food. For the peanut butter study, the system was compared to the U.S. Food and Drug Administration's Bacteriological Analytical Manual procedures using an unpaired-study design. No significant difference in performance was observed between the MicroSEQ Salmonella spp. detection system and the corresponding reference methods for all 11 food matrixes. The MicroSEQ system detected all Salmonella strains tested, while showing good discrimination against detection of an exclusivity panel of 30 strains, with high accuracy.     

A theoretical introduction to “Combinatory SYBR®Green qPCR Screening”, a matrix-based approach for the detection of materials derived from genetically modified plants

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. “Combinatory qPCR SYBR®Green screening” (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBR®GREEN qPCR analysis based on four values: the C _t- and T _m values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, “Prime number tracing”, matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBR®GREEN qPCR methods and through application of a newly developed “prime number”-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product.     

Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers’ perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

Towards future reference systems for GM analysis

Despite the fact that the measurement unit for the quantification of GMOs in food and feed products has not yet been unambiguously agreed upon in Europe, international trade requires reliable GMO analysis measuring comparably the GMO content of products. The two reference systems, based either on mass fractions or on copy number ratios, and their metrological traceability chains are presented and discussed. It is concluded that, properly established and expressed, measurement results in copy number ratios can provide a metrologically sound reference system. In this case, certified reference materials used for calibration and quality control can be independent of each other and the uncertainty derived from calibration can correctly be included in the overall uncertainty of the GMO measurement. However, further efforts are required to establish this metrological system.     

Bioanalytical methods for food contaminant analysis

Foods are complex mixtures of lipids, carbohydrates, proteins, vitamins, organic compounds, and other naturally occurring substances. Sometimes added to this mixture are residues of pesticides, veterinary and human drugs, microbial toxins, preservatives, contaminants from food processing and packaging, and other residues. This milieu of compounds can pose difficulties in the analysis of food contaminants. There is an expanding need for rapid and cost-effective residue methods for difficult food matrixes to safeguard our food supply. Bioanalytical methods are established for many food contaminants such as mycotoxins and are the method of choice for many food allergens. Bioanalytical methods are often more cost-effective and sensitive than instrumental procedures. Recent developments in bioanalytical methods may provide more applications for their use in food analysis.     

Validation of two commercial lateral flow devices for the detection of peanut proteins in cookies: interlaboratory study

Results are reported for an interlaboratory validation study of 2 commercially available Iateral flow devices (dipstick tests) designed to detect peanut residues in food matrixes. The test samples used in this study were cookies containing peanuts at 7 different concentrations in the range of 0-30 mg peanuts/kg food matrix. The test samples with sufficient and proven homogeneity were prepared in our laboratory. The analyses of the samples (5 times per level by each laboratory) were performed by 18 laboratories worldwide which submitted a total of 1260 analytical results. One laboratory was found to be an outlier for one of the test kits. In general, both test kits performed well. However, some false-negative results were reported for all matrixes containing < 21 mg peanuts/kg cookie. It must be stressed that the test kits were challenged beyond their cut-off limits (> or = 5 mg/kg, depending on the food matrix). One test kit showed fewer false-negative results, but it led to some false-positive results for the blank materials. The sensitivity of the dipstick tests approaches that achieved with enzyme-linked immunosorbent assays.     

Development of a potential reference material for evaluating antioxidant activity

Phenolic phytochemical are known to perform several functions ranging from phytoprotectants, to protecting lipids in food products, to antioxidant activity in animals and humans. The need for a common standard mixture containing multiple phenolic phytochemicals is critical for the development of a robust validation assay for accurately quantifying antioxidant activity in various matrixes. Different research groups have used A wide array of single purified reference phenolic compounds in this regard. A 5 compound mixture (caffeic acid, morin hydrate, hesperetin, catechin hydrate, and epigallocatechin gallate) containing phenolic compounds from 4 subgroups (phenolic acid, flavone, flavanone, and flavan-3-ol) was prepared. The mixture was assayed for stability evaluation by high-performance liquid chromatography (HPLC) using a diode array detection procedure for a 3 month time interval. HPLC analysis confirmed that there was no significant interaction between different components of the mixture. The among-sample relative standard deviation (RSD) of all 5 phenolic compounds, as well as the total HPLC area, was < 1%. The RSD due to instrument variation was < 2% and the total RSD among-days was < 5%. These results unambiguously suggest that the sample was stable for a 3 month time interval in an amber vial stored in a refrigerator below 5 degrees C. This mixture is currently being used for the single-laboratory validation study for the assay of total phenolic content by the Folin-Ciocalteu method and the antioxidant capacity by oxygen radical absorbing capacity procedure.     

Development and validation of a method for the quantification of milk proteins in food products based on liquid chromatography with mass spectrometric detection

The protection of allergic consumers is crucial to the food industry. Therefore, accurate methods for the detection of food allergens are required. Targeted detection of selected molecules by MS combines high selectivity with accurate quantification. A confirmatory method based on LC/selected reaction monitoring (SRM)-MS/MS was established and validated for the quantification of milk traces in food. Tryptic peptides of the major milk proteins beta-lactoglobulin, beta-casein, alphaS2-casein, and K-casein were selected as quantitative markers. Precise quantification was achieved using internal standard peptides containing isotopically labeled amino acids. For each peptide, qualifier and quantifier fragments were selected according to Commission Decision 2002/657/EC. A simple sample preparation method was established without immunoaffinity or SPE enrichment steps for food matrixes containing different amounts of protein, such as baby food, breakfast cereals, infant formula, and cereals. Intermediate reproducibility, repeatability, accuracy, and measurement uncertainty were determined for each matrix. LOD values of 0.2-0.5 mg/kg, e.g., for beta-lactoglobulin, were comparable to those obtained with ELISA kits. An LOQ of approximately 5 mg/kg, expressed as mass fraction skim milk powder, was validated in protein-rich infant cereals. The obtained validation data show that the described LC/SRM-MS/MS approach can serve as a confirmatory method for the determination of milk traces in selected food matrixes.