Marco Mazzara

International Collaborative Study of the Endogenous Reference Gene, Sucrose Phosphate Synthase (SPS), Used for Qualitative and Quantitative Analysis of Genetically Modified Rice

One rice ( Oryza sativa ) gene, sucrose phosphate synthase (SPS), has been proven to be a suitable endogenous reference gene for genetically modified (GM) rice detection in a previous study. Herein are the reported results of an international collaborative ring trial for validation of the SPS gene as an endogenous reference gene and its optimized qualitative and quantitative polymerase chain reaction (PCR) systems. A total of 12 genetically modified organism (GMO) detection laboratories from seven countries participated in the ring trial and returned their results. The validated results confirmed the species specificity of the method through testing 10 plant genomic DNAs, low heterogeneity, and a stable single-copy number of the rice SPS gene among 7 indica varieties and 5 japonica varieties. The SPS qualitative PCR assay was validated with a limit of detection (LOD) of 0.1%, which corresponded to about 230 copies of haploid rice genomic DNA, while the limit of quantification (LOQ) for the quantitative PCR system was about 23 copies of haploid rice genomic DNA, with acceptable PCR efficiency and linearity. Furthermore, the bias between the test and true values of eight blind samples ranged from 5.22 to 26.53%. Thus, we believe that the SPS gene is suitable for use as an endogenous reference gene for the identification and quantification of GM rice and its derivates.

Use of pJANUS-02-001 as a calibrator plasmid for Roundup Ready soybean event GTS-40-3-2 detection: an interlaboratory trial assessment.

Owing to the labelling requirements of food and feed products containing materials derived from genetically modified organisms, quantitative detection methods have to be developed for this purpose, including the necessary certified reference materials and calibrator standards. To date, for most genetically modified organisms authorized in the European Union, certified reference materials derived from seed powders are being developed. Here, an assessment has been made on the feasibility of using plasmid DNA as an alternative calibrator for the quantitative detection of genetically modified organisms. For this, a dual-target plasmid, designated as pJANUS™-02-001, comprising part of a junction region of genetically modified soybean event GTS-40-3-2 and the endogenous soybean-specific lectin gene was constructed. The dynamic range, efficiency and limit of detection for the soybean event GTS-40-3-2 real-time quantitative polymerase chain reaction (Q-PCR) system described by Terry et al. (J AOAC Int 85(4):938–944, 2002) were shown to be similar for in house produced homozygous genomic DNA from leaf tissue of soybean event GTS-40-3-2 and for plasmid pJANUS™-02-001 DNA backgrounds. The performance of this real-time Q-PCR system using both types of DNA templates as calibrator standards in quantitative DNA analysis was further assessed in an interlaboratory trial. Statistical analysis and fuzzy-logic-based interpretation were performed on critical method parameters (as defined by the European Network of GMO Laboratories and the Community Reference Laboratory for GM Food and Feed guidelines) and demonstrated that the plasmid pJANUS™-02-001 DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2 in food and feed products.

Inter-laboratory Testing of GMO Detection by Combinatory SYBR®Green PCR Screening (CoSYPS)

Combinatory SYBR®Green real-time PCR Screening (CoSYPS) is an efficient, sensitive approach for detecting complex targets such as genetically modified organisms (GMOs) in food and feed products. GMO analysis for legal purposes has become increasingly complex and costly due to the diversity in recombinant targets present in the different GMOs. For this reason, screening for the presence of GMOs is in general the first step in the detection of GM material in a product. CoSYPS allows detecting the large majority of globally commercial GMOs using SYBR®Green real-time PCR methods for six GM targets (P35S, Tnos, CryIAb, CP4-EPSPS, PAT and BAR) combined with species-specific PCR methods (e.g., maize, soy, rapeseed). Here, the results of an inter-laboratory trial on seven samples with different GMO mixtures at different levels are presented. In total, 13 laboratories participated in the trial and the currently most frequently used PCR analysis platforms are represented. The inter-laboratory study clearly demonstrates that PCR methods used in CoSYPS form a very robust GMO screening system. Sensitivity, specificity, positive and negative predictive values are for all PCR methods higher than 95xa0% for all samples. Together, these results show that the SYBR®Green real-time PCR methods used in CoSYPS are effectively applicable to different PCR platforms and amendable to configuration into a sensitive high-throughput GMO screening and decision support tool.

New SYBR®Green methods targeting promoter sequences used for screening of several GM events pending for authorisation in Europe

Seen the growing number of genetically modified (GM) crops being developed, the need for cost- and time-effective detection methods is increasing to enable continuing the necessary effective control on food and feed products. This need can be achieved by performing an intensive screening combined with decision support tools like the CoSYPS matrix which permits reducing the number of events to be identified. To allow an extra covering power of the CoSYPS and to be able to include new EU-authorised GM events, two new SYBR®Green real-time PCR (qPCR) methods targeting two promoter sequences (pNOS and pFMV) were developed. These methods were validated using acceptance parameters such as the specificity, sensitivity and repeatability. In addition, the methods were transferred to a second laboratory, namely the Institute for Health and Consumer Protection, to test the reproducibility. Furthermore, the applicability and practicability of the methods were tested by using proficiency test samples. The two methods allow a specific and sensitive detection of the targets in food and feed samples and can be used efficiently in different laboratories.

Testing the interaction between analytical modules: an example with Roundup Ready®soybean line GTS 40-3-2

BackgroundThe modular approach to analysis of genetically modified organisms (GMOs) relies on the independence of the modules combined (i.e. DNA extraction and GM quantification). The validity of this assumption has to be proved on the basis of specific performance criteria.ResultsAn experiment was conducted using, as a reference, the validated quantitative real-time polymerase chain reaction (PCR) module for detection of glyphosate-tolerant Roundup Ready® GM soybean (RRS). Different DNA extraction modules (CTAB, Wizard and Dellaporta), were used to extract DNA from different food/feed matrices (feed, biscuit and certified reference material [CRM 1%]) containing the target of the real-time PCR module used for validation. Purity and structural integrity (absence of inhibition) were used as basic criteria that a DNA extraction module must satisfy in order to provide suitable template DNA for quantitative real-time (RT) PCR-based GMO analysis. When performance criteria were applied (removal of non-compliant DNA extracts), the independence of GMO quantification from the extraction method and matrix was statistically proved, except in the case of Wizard applied to biscuit. A fuzzy logic-based procedure also confirmed the relatively poor performance of the Wizard/biscuit combination.ConclusionsFor RRS, this study recognises that modularity can be generally accepted, with the limitation of avoiding combining highly processed material (i.e. biscuit) with a magnetic-beads system (i.e. Wizard).

Combinatory SYBR ® Green Real-Time PCR Screening Approach for Tracing Materials Derived from Genetically Modified Rice

Two real-time PCR approaches for the detection of genetically modified (GM) rice were tested: (1) a combination of SYBR® Green real-time PCR methods detecting the 35S promoter (P-35S) of Cauliflower Mosaic Virus, the nopaline synthase terminator (T-nos) of Agrobacterium tumefaciens and the Bacillus thuringiensis (Bt) CryIAb/Ac toxins and (2) a P-35S/T-nos duplex TaqMan® real-time PCR system. Both systems correctly recognized their respective targets in control samples of Bt63, Kefeng6 and KMD1 insect-resistant and LLRice62 and LLRice601 herbicide-resistant rice. Due to its lesser specificity but broader genetically modified organism (GMO) coverage capacity, the SYBR® Green real-time PCR approach was further tested in more detail. Melting curve, capillary and agarose gel electrophoresis analyses indicated that the P-35S, T-nos and CryIAb/Ac targets in the GM rice events are similar to the corresponding targets present in many known GMOs. High-resolution melting analysis showed that the CryIAb/Ac targets of the GM rice events Bt63 and Kefeng6 matched best the corresponding Bt11 CryIAb sequence. Digital PCR analysis on genomic DNA from the GM rice Bt63 and Kefeng6 events indicated that both GMO contained multiple inserts. Sensitivity tests showed that all SYBR® Green real-time PCR methods could detect their targets at less than an estimated five copies per reaction. Finally, it was shown that these SYBR® Green real-time PCR methods could detect low levels of their targets in rice consignments originating from China. Together, these results demonstrated that a ‘P-35S and T-nos and CryIAb/Ac’ combinatory SYBR® Green real-time PCR screening is highly suited to trace the respective targets including the possible presence of Bt63, Kefeng6 and KMD1 GM rice materials in food products.

Kernel Lot Distribution Assessment (KeLDA): a Comparative Study of Protein and DNA-Based Detection Methods for GMO Testing

Monitoring of market products for detection of genetically modified organisms (GMO) is needed to comply with legislation in force in many regions of the world, to enforce traceability and to allow official control along the production and the distribution chains. This objective can be more easily achieved if reliable, time and cost-effective analytical methods are available. A GMO can be detected using either DNA-based or protein-based methods; both present advantages and disadvantages. The objective of this work was to assess the performance of a protein-based (lateral flow strips—LFT) and of a DNA-based (polymerase chain reaction—PCR) detection method for GMO analysis. One thousand five hundred samples of soybean, deriving from the sampling of 15 independent bulk lots in large shipments, were analysed to assess and compare the performance of the analytical methods and evaluate their suitability for GMO testing. Several indicators were used to compare the performance of the methods, including the percentage correlation between the PCR and LFT results. The GMO content of the samples ranged from 0 up to 100xa0%, allowing a full assessment of both analytical approaches with respect to all possible GMO content scenarios. The study revealed a very similar performance of the two methodologies, with low false-negative and false-positive results, and a very satisfactory capacity of both methods in detecting low amounts of target. While determining the fitness for purpose of both analytical approaches, this study also underlines the importance of alternative method characteristics, like costs and time.

Development, Optimization, and Single Laboratory Validation of an Event-Specific Real-Time PCR Method for the Detection and Quantification of Golden Rice 2 Using a Novel Taxon-Specific Assay

In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3 insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

International Ring Trial for the Validation of an Event-Specific Golden Rice 2 Quantitative Real-Time Polymerase Chain Reaction Method

This article describes the international validation of the quantitative real-time polymerase chain reaction (PCR) detection method for Golden Rice 2. The method consists of a taxon-specific assay amplifying a fragment of rice Phospholipase D α2 gene, and an event-specific assay designed on the 3 junction between transgenic insert and plant DNA. We validated the two assays independently, with absolute quantification, and in combination, with relative quantification, on DNA samples prepared in haploid genome equivalents. We assessed trueness, precision, efficiency, and linearity of the two assays, and the results demonstrate that both the assays independently assessed and the entire method fulfill European and international requirements for methods for genetically modified organism (GMO) testing, within the dynamic range tested. The homogeneity of the results of the collaborative trial between Europe and Asia is a good indicator of the robustness of the method.

Testing the Robustness of Validated Methods for Quantitative Detection of GMOs Across qPCR Instruments

The number of real-time polymerase chain reaction (qPCR) instruments available has greatly increased over recent years. However, little information is available on the performance of validated quantitative methods when tested in different instruments. A study has been designed to evaluate the robustness of three validated methods for genetically modified organisms (GMO) quantification across six real-time platforms from four different suppliers. The performance of three validated methods for event-specific detection of Bt11 maize, DAS 59122 maize and MON 89788 soybean was evaluated on six qPCR platforms (ABI 7900 HT, ABI Prism® 7700 and ABI 7500 from Applied Biosystems; LightCycler® 480 from Roche; Mx3005P® from Stratagene; and iQ™5 from Bio-Rad). Method performance criteria were compared against European Network of GMO Laboratories method performance requirements for analytical methods of GMO testing. The latter comparison indicates that the criteria are fulfilled for most of the platforms and levels of GMO concentrations, though with minor exceptions. The analysis of variance (one-way) indicated that the quantification of GMOs was affected by the platforms, which did not respond consistently across GM levels. A two-way analysis of variance confirmed that there were significant interactions between platforms and GM levels. The pairwise comparison of the platforms performance indicated that some deviate significantly from others, though differences between methods were observed.